science.adg9091

Supplementary

Materials

for

The mechanism of the phage

encoded protein antibioti

c from



X174

Anna K. Orta

et al

Corresponding author

William M. Clemons Jr., clemons@caltech.edu

Sci

ence

adg9091

(

202

)

DOI:

10.1126/sci

ence

adg9091

The

PDF

file

includes:

Figs. S1 to S14

Table S1

References

Other Supplementary Material for this manuscript includes the following:

MDAR Reproducibility Checklist

Movie S1

Materials and Methods

xpression

of EcMraY,

protein E

and EcSlyD

∆

slyD

BL21(DE3) competent cells were co

transformed with pET22b

SlyD

154

and either

pRSFDuet

MraY

ID21

or pRSFDuet

MraY

X174

and plated in LB

agar containing 35

g/ml Kanamycin and 100

g/mL Ampicillin. Our pET22b

SlyD

154

construct expresses

E. coli

SlyD, modified by the removal of the flexible C

terminus. The pRSFDuet

MraY

ID21

plasmid

contains the ID21 isoform of

protein E

along with a wild

type

MraY to prevent cell lysis from

the overexpression of

protein E

. Cells were grown in 2xYT media at 37

◦

C, 225 r.p.m., and induced

at an OD

600

of 0.9 with 0.4mM IPTG at 18

◦

C overnight. The culture was harvested by

centrifugation for

10 minutes at 9,000

, 4

◦

C then frozen or used immediately for purification.

Purification of the YES complex

The cells were resuspended in lysis buffer (20mM Tris

HCl pH 7.5, 300 mM NaCl, 10% Glycerol,

5mM

mercaptoethanol

(

)

, 0.1mM PMSF, 0.1mM

Benzamidine) and homogenized using a

110L

microfluidizer (Microfluidics). The lysate was cleared by a

minute

centrifugation at

a speed of 22,000

. The supernatant was then centrifugated at 167,424

and the resulting

membrane pellet was then solubili

zed in the extraction buffer (10 mM HEPES pH 7.5, 300 mM

NaCl, 5% Glycerol, 5mM

, 0.1mM phenylmethylsulfonyl fluoride

(PMSF)

, 0.1mM

benzamidine, 10 mM imidazole and 1% dodecyl 4

glucopyranosyl

glucopyranoside

(DDM)) After allowing for extracti

on for 1.5 hours at 4

◦

C, the solution was centrifuged at

167,424

for 30 minutes and the remaining lysate was mixed with 1mL NiNTA resin (Qiagen,

Alameda, CA) then nutated at 4

◦

C for two hours. This solution was loaded onto a gravity column

and then washe

d with five column volumes of wash buffer (10 mM HEPES pH 7.5, 150 mM

NaCl, 5% glycerol, 5mM

, & 0.03% DDM) with 10mM imidazole followed by five column

volumes of wash buffer with 30 mM imidazole. The YES complex was eluted in 20mL of wash

buffer contai

ning 200 mM imidazole. The final purification step was SEC (Superdex 200 5/150

GL,

Millipore

Sigma) in 10mM HEPES pH 7.5, 75 mM NaCl, 5% Glycerol, 5mM

and

0.03% DDM. Fractions were assessed by SDS

PAGE and directly used for cryo

sample

preparation.

expression of EcMraY and

protein E

in various SlyD backgrounds

The pRSFDuet

MraY and pRSFDuet

Epos

MraY expression vectors were transformed

into BL21

Star cells (Novagen). Similarly, the pRSFDuet

E(C

term)

SlyD

154

was transformed

into SlyD

knocko

ut cells. The cultures were grown at 37

◦

C to an OD

600

0.8 and induced with 1

mM IPTG. Induced cultures were grown for 3 hours followed by harvesting by centrifugation at

9,000

for 20 min. Cell pellets were resuspended in lysis buffer and lysed by sonica

tion. The

lysate was then cleared by centrifugation at 22,000

, followed by a second centrifugation at

234,78

for 1 hour to isolate the membrane fraction. The complex was extracted in 20 mM Tris

HCl pH 7.5, 300 mM NaCl, 10% Glycerol, 10 mM Imidazole, an

d 1% n

Decyl

Maltoside (DM)

and incubated at 4

◦

C for 1.5 hours. The debris was cleared by centrifugation at 234,788

for 30

min. The sample was incubated with 1 mL NiNTA resin for 1 hour, followed by a wash with 50

column volumes lysis buffer with 30mM

Imidazole. The

protein

E complexes were similarly

eluted in 300mM Imidazole. The elutions were concentrated and further purified by size exclusion

chromatography (Superdex 200 5/150 GL,

Millipore

Sigma).

Lysis assays of WT

protein E

X174 and ID21

LEMO DE3 competent cells were transformed with a pRSF

Duet vector either empty, with

protein

X174

, or

protein E

ID21

. Cultures were grown to an OD

600

of 0.2 and inoculated into a

Corning 96

well Clear Flat Bottom plates in 100

L triplicate aliquots and in

duced as described

previously. Cultures were incubated at 37C with orbital shaking at 220rpm using an Infinite M

Nano+ (Tecan, Switzerland). Readings were taken in 5

minute intervals for 90 minutes.

Lysis assay for

protein E

constructs

LEMO DE3 competent cells (New England Biolabs, MA, USA) were transformed with a

pRSFDuet vector either empty, with C

terminally FLAG tagged

protein

X174

variants (WT,

P21A, K46A). The lysis assays were performed in

three

biological

replicates

as previous

described

(

)

. Absorbance readings were recorded in

minute intervals for 1 hour and 30

minutes. Manual readings were taken using a Biowave Cell Density Meter CO8000. The values

were plotted using GraphPad Prism version 9.1.1 for

macOS.

Lysis assays based on SlyD variants

∆

slyD

(

)

cells were transformed with either a control empty pRSF

Duet vector or pRSFDuet

Protein

X174

and either pET22b

SlyD, pET22b

SlyD

154

, pET22b

SlyD Y68K, or pET22b

Thermus thermop

hilus

SlyD. Cultures were grown in 2xYT media at 37

◦

C and induced with

0.4mM IPTG once at an OD

600

of 0.2. Absorbance measurements were manually recorded in 5

minute intervals for 70 minutes. Similarly,

∆

slyD

cells were transformed with either a control

empty pRSF

Duet vector or pRSF

Duet

Protein

ID21

either alone, with pET22b

SlyD, or with

pET22b

SlyD

154

and induced with 0.4mM IPTG. Readings were recorded using an Infinite M

Nano+ plate reader as described a

bove.

Sample preparation for

ryoEM

The YES complex was diluted to 5.0 mg/mL in 10 mM HEPES p

7.5, 75 mM NaCl, 5%

Glycerol, 5mM

ME and 0.03% DDM

. Additionally, the YES

ID21

sample was

supplemented with

1mM

E. coli

total lipid extract (Avanti Polar Lipids, 100600P). Quantifoil holey carbon films

R1.2/1.3 300 Mesh, Copper (Quantifoil, Micro Tools GmbH) grids were glow discharged with a

minute 20

plasma current using a

Pelco easiGlow, Emitech K100X. Grids were prepa

red

using a Vitrobot (FEI Vitrobot Mark v4 x2, Mark v3) by

applying

L of sample onto the grid

followed by a 3.5 second blot using a +8

blot

force and plunge frozen into liquid ethane.

Data acquisition and analysis

The grids were imaged in a 300 kV cryo

TEM microscope equipped with a Gatan K3 6k x 4k

direct electron detector and a Gatan Energy Filter (slit width 20eV) in super

resolution mode

using Serial EM.

Datasets

were collected at a 105k magnification with a pixel size of 0.416

/pixel. Movies with 4

0 frames were recorded with a total

exposure

dose of 60 e

and a defocus

range of

1.0 to

For the YES

ID21

complex, a

total of 12,070 movies were recorded.

Movies were normalized by gain reference and motion corrected using the patch motion

correction built in function in cryosparc (v3.3.2) with a two

fold bin that resulted in a pixel size

of 0.832

/pixel

(

)

. The contrast transfer function (CTF) was estima

ted using CTFFIND4

(

)

Micrographs were manually curated, and low

quality images were removed for further analyses.

A total of 2,462,335 particles were obtained followed by the generation of 6 ab

initio models. Out

of the 6 models, two models are selecte

d for classification

into

“

good

” and

“

trash” volumes.

All

particles were then sorted in these two volumes through heterogeneous refinement using particles

extracted with a 4x bin, which produced 6,589,696 good particles. Heterogeneous refinement was

used i

n an iterative manner to sort the particles into the 5 volumes (4 good and 1 trash). The

1,151,777 good particles were used for non

uniform homogeneous refinement to generate a

higher resolution volume. The particles were then extracted with a 3x bin and s

orted into 4

iterations of the higher resolution volume and 1 trash volume. Iterative rounds of heterogeneous

refinement at 3x bin produced 935,754 particles. Particles were then extracted in a 2x bin and

heterogeneously refined into either

high

or low

solution

volumes. At this point, discerning

features in the soluble region of the model were used to select the most complete volumes. The

volumes were individually refined through non

uniform refinement and the particles that

composed the volumes with mos

t complete and highest resolution were used. A total of 122,452

particles were used for the most complete model obtained upon non

uniform refinement.

The

FSC

masked resolution was 3.

Å, while the unmasked resolution

was 3.9Å

The half

maps were

then used for post

processing through DeepEMhancer

(

)

with the high

resolution model

selected for our most complete density map. Post

processing through DeepEMhancer removed

the micelle and improved the features on the soluble portions

of the map, however the lipid

densities were also removed. The lipid densities described in this work are those

of the YES

ID21

map

before post

processing.

Notably, the dimer

interface lipid density was also present in the

YES

X174

density map without the

supplementation of

coli

lipid extract.

Figure 1D uses the

densities befor

e post

processing for the MraY dimer

, micelle and lipid densities

, and the

DeepEMhancer

post

processed map for

protein E and SlyD.

Supplemental figures S9 and S

were

made with

the

map before DeepEMhancer sharpening.

The YES

X174

complex dataset was

processed in this same manner

. A total of 10,798 movies were recorded. The model for the

YES

ID21

complex was then used as a template for template picking

, from which 1,516,

368

partic

les were

picked and

curated.

Following gradual un

binning

and sorting into good and trash

volumes,

155,270 particles were used for the final

iteration of non

uniform refinement

The

local

resolution of both maps was performed on cryosparc (v3.3.2).

The half

maps

were then post

processed using DeepEMhancer

as described previously.

Model building

For starting

model

s we used

the

Aquifex

aeolicus

bound

structure (PDBID:4J72

(

)

for

MraY and

the

E. coli

NMR

structure (

PDBID:2K8I

(

)

for

SlyD

which were fit

using

phenix.dock.

SlyD was then split into its two domains, IF and FKBP

at residues Y68 and

G127

Protein E was modeled

novo

to residue P65

using Coot 0.8.9.2. The structure

of the

MraY,

protein E

, and

SlyD

domain

ere

refined using phenix.real space refinement and

ISOLDE 1.6,

and validated with PHENIX

1.19.2.

After the refinements of

MraY,

protein

and

the

SlyD

domain

were completed, the FKBP domains were docked

into density

using

ChimeraX.

The complete

YES complex

structure was then refined

using PHENIX

1.19.2 and

ISOLDE

1.6.

RMSDs were calculated using ChimeraX Matchmaker chain alignment. Structure

figures were made

using ChimeraX and sequence alignments using Jalview

and ClustalW

(

–

)

Table S1

Cryo

EM data collection,

refinement,

and validation statistics

YES

ID21

YES

ΦX174

(EMDB

29641)

(EMDB

29642)

(PDB 8G01)

(PDB 8G02)

Data collection and processing

Microscope

FEI Titan Krios

Magnification

105,000

Voltage (kV)

300

Electron exposure (e

–

/Å

)

Defocus range (μm)

1 to

2.5

1 to

2.5

Pixel size (Å)

0.832

Symmetry imposed

Initial particle images

(no.)

11,700,795

1,516,368

Final particle images (no.)

122,452

155,270

Map resolution (Å)

3.47

3.6

FSC threshold: 0.143

Refinement

Software

PHENIX 1.19.2

Initial model used (PDB code)

4J72, 2K8I

8G01

Resolution of unmasked

reconstructions (Å, FSC=0.5)

3.72

3.8

Resolution of masked

reconstructions (Å, FSC=0.5)

3.72

3.79

Correlation coefficient (

mask

)

0.71

0.69

Map sharpening

factor (Å

)

142.4

148.3

Model composition

Atoms (Hydrogens)

18153 (9214)

18198 (9232)

Protein residues

1148

Ligands

factors (Å

)

min/max/mean

Protein

60.55/203.29/93.87

48.57/311.57/93.33

Ligand

R.m.s. deviations

Bond lengths (Å)

0.002 (0)

0.003 (0)

Bond angles (°)

0.436(0)

0.636(2)

Validation

MolProbity score

1.12

1.53

Clashscore

3.31

10.33

Poor rotamers (%)

0.1

Ramachandran plot

Favored (%)

99.03

98.15

Allowed (%)

0.97

1.85

Disallowed (%)

Fig. S1. Broad comparison of

protein E

isoforms from

Bullavirinae

(

)

Similar to Fig.

, sequence alignment

protein

E species from UniRef90

(

)

using ClustalW. Helices are indicated above the alignment. The TMD and E

term are indicated. ΦX174 and ID21 are highlighted.

(

)

Phylogenetic tree of

protein E

isoforms with branch

lengths annotated.

Fig. S2. Stable complex formation of SlyD with

protein E

(

)

expressed Epos (E

ΦX174

R3H, L19F) with

MraY in a wild

type

E. coli

background purified by SEC and the corresponding

SDS

PAGE. Protein bands are

labeled. (

)

ClustalW s

equence alignment of

E. coli

SlyD and

Thermus thermophilus

SlyD

. The residues composing

the IF domain are underlined and labeled.

Asterisks (*) denote contact residues between

SlyD and protein E.

Arrow

points to the truncation for

SlyD

154

(

)

Lysis assay in a ∆

slyD

background.

Either empty vector (

∗

protein

ΦX174

alone (

■

), or

protein

ΦX174

with either

SlyD (

▼

SlyD

154

(

●

SlyD Y68K (

◆

), or

SlyD (

▲

These are

representative from single

samples.

(

)

Similar to (

), lysis assay in a ∆

slyD

background with empty vector (

∗

protein

ID21

alone (

■

) or

protein

ID21

with either

SlyD (

▼

) or

SlyD

154

(

●

Error bars

represent the

standard

deviation

derived from

(

)

SDS

PAGE of purified

protein

E C

term from either ID21 or

3 co

expressed with

SlyD

154

(

) Structural alignment of the YES complex

SlyD

IF domain

(gray)

and the NMR

SlyD structure

(

PDBID:2K8I

, viridis)

(

)

The

complete structure of

the YES

SlyD

molecule is shown at the top left, with

FKBP and IF domains labeled respectively. (

) As in (

)

, structural alignment of

YES

SlyD

(gray) to

SlyD

(PDBID:2K8I)

(

)

aligned to the FKBP domain. The viridis

gradient shows the N

terminus in purple and C

terminus

in yellow

(

)

(

) Co

expressed Epos with

MraY in ∆

slyD

background purified by SEC and the corresponding

SDS

PAGE. Arrow highlights the fraction run on the gel.

Fig. S3. CryoEM

processing pipeline for YES

ID21

complex.

Processing was done using cryosparc v3.2.0. The order

of processing follows the arrows. The number of particles after each step is shown. Local resolution is shown in a

Plasma color scheme ranging from 3.0

(yellow

) to 3.8

(purple) resolution. Overall GSFSC resolution was

determined on

cryosparc

(

v3.2.0

)

Final model was sharpened using DeepEMhancer

(

)

Fig. S4. CryoEM processing pipeline for YES

ΦX174

complex.

Processing was done using cryosparc

(

v3.2.0

)

. The

order of processing follows the arrows. The number of particles after each step is shown. Local resolution is shown in

a Plasma color scheme ranging from 3.0

(yellow) to 3.

8Å

(purple) resolution. Overall GSFSC resolution was

determined

cryosparc

(

v3.2.

)

Fig. S5. Comparison of the YES

ID21

complex and the YES

ΦX174

complex.

(

)

Similar to Fig.

for protein density

of the YES

ID21

complex.

(

)

Density maps (blue mesh) for

protein

ID21

with protein in stick representation

highlighting the regions that contain the TMD.

(

)

the amphipathic cytoplasmic helix.

(

–

)

, similar to (

) except

for the YES

ΦX174

complex. Here protein E is shown in orange.

(

)

Local resolution

map for

the protein

ID21

density

before

eepEMhancer sharpening

using the p

lasma color scheme ranging from 3.

(yellow) to

4.0

Å (purple)

resolution.

(

)

The N

terminal interactions of

protein

ID21

with

MraY. Proteins are shown in cartoon with relevant

side chains as stic

ks.

(

)

As in (

), for

protein

ΦX174

. In

(

, &

)

residues that vary between the two isoforms

are labeled. Residues I28, P29, and M50 are labeled for orientation.

Fig. S6. Symmetry in the YES complex.

(

)

Overlay of a symmetry imposed, 180

◦

rotation, of the YES

ID21

complex.

The proteins are shown as cartoons in either cyan or pink.

(

)

ribbon of the two structurally aligned

protein

ID21

monomers (A: Viridis

(

)

, B: yellow). Symmetry is broken after res

idue S43. The inset shows the orientation relative

to A.

(

)

ribbon of the two structurally aligned MraY monomers (A: Viridis, B: cyan). The inset shows the

orientation relative to

(

)

as in (

). The NTH and Loop 1

2 are labeled.

(

)

Viewed from the cytoplasm, cartoon

models of SlyDs from the aligned YES complex in (

) highlighting the change in orientation.

(

)

ribbon of the

structurally aligned SlyD monomers (A: Viridis, B: light purple).

Fig. S7. Multiple sequence alignment o

f representative MraY homologs.

Structure based multiple sequence

alignment using Promals3D with ClustalW coloring for residues. Species were selected by using the phylogenetic tree

selecting from the cutoff shown in

ig. S11. Uniprot ID of

MraY homologs

(in order): O26830, Q9WY77,

0BTE

Q03521, Q182Y8, P9WMW7, O25235, A8UQI5,

O66465

Q88N79, Q8E9P5, B0BRH4, Q9KPG4, A6T4N0,

P0A6W3. Secondary structure based

MraY from the YES complex structure is shown below the sequences, TMDs

are colored as in Fig.

. Side chains in MraY that contact

protein E

are labeled with a (*).

Fig. S8. Sequence variability for the coding region of Proteins E from ΦX174, ID21, and G4.

(

)

The DNA

sequence of gene E and the overlapping gene D in the bacteriophages ΦX174, ID21, and G4. Nucleotide differences

in the sequences are highlighted in dark pink. Translated protein sequence for ΦX174

protein

E is shown above the

DNA sequences and Pro

tein D. Amino acids are colored from Benchling (Biology Software,2022). For ID21 or G4,

only residues that differ from ΦX174 in the two proteins are shown.

(

)

Left, the structure of the ΦX174 pro

virus

capsid (PDB:1CD3

(

)

) with proteins shown as cartoon

and colored to highlight the symmetry related Protein Ds.

Right, a zoomed in view of one Protein D monomer colored either light blue or yellow for the region that overlaps

with

protein E

in yellow. The positions where sequence changes in

protein E

result

in sequence changes in Protein D

are shown as sticks.

Fig. S9. Structural features of

MraY.

(

)

Cartoon representation of

MraY colored in Viridis

(

)

monomeric unit A and B. Foreground transmembrane helices are labeled 1

10. Loops 1

2, 9

10, and the NTH are

labeled for reference.

(

)

Monomer of MraY viewed from the active site cleft (left) or the dimer interface (right).

(

)

Cartoon representation o

f the N

terminal helix of MraY with N

terminal and TM2

side chains

shown as sticks.

Inset

(right) shows

the

backbone

hydrogen bonding between

the

TM2 and NT

. Arrow highlights the N

terminus

Density

map is shown as a blue mesh.

(

)

Periplasmic (left) and cytoplasmic (right) view of the MraY monomer.

Transmembrane helices are labeled.

(

)

Cartoon representation with stick side chains of TM10. Densities shown as a

blue mesh.

(

)

Stick model of Loop 9

10 with density (blue mesh) orient

ed as box in (

(

)

As (

) for Loop 1

Fig. S10. Structural alignment of experimental MraY structures.

For each panel, viewed from the active site cleft

and from the cytoplasm.

(

)

MraY is shown in Viridis

and transmembrane domains are labeled. Protein E

ID21

shown in gray.

(

)

Uninhibited

MraY (PDB

:4J72

(

)

) in orange.

(

)

Carbacaprazamycin inhibited

MraY

(PDB:6OYH

(

)

) in pink.

(

)

Tunicamycin inhibited

MraY (PDB

:5JNQ

(

)

) in green.

Fig. S11

. Conservation of the N

terminal helix stacking.

(

)

Phylogenetic tree of MraY from representative

bacterial species. Species demonstrated to from ghosts when

protein E

is expressed are highlighted by an asterisk (*).

Selected species are highlighted in yellow.

(

)

MraY cryoEM structure colored in Viridis with the N

terminal

helices highlighted.

(

)

AlphaFold prediction of MraY structures from Gram

negative species ori

ented and shown as

in (

(

)

AlphaFold prediction of MraY structures from Gram

positive species oriented and shown as

in (A)

Fig. S12. Likely lipid densities bound in the YES complex.

(

)

Cartoon representation of the YES

(ID21)

complex

(gray) with

likely lipid densities in orange. Purple density is possible C

P. Inset shows possible phospholipid

densities.

(

)

As in a with a 90

◦

rotation viewed towards the active site cleft.

(

)

Putative pocket

(

)

for binding the

phosphate of C

P. Putative bindi

ng residues and the modeled C

P (purple) are shown as sticks.

Fig. S13. Structural variability in the YES complex.

variability analysis of the YES complex. Movement is

visualized by highlighting the first (

) and last frame (