Supplementary Table
1:
Cryo
-
EM data collection, refinement
,
and validation statistics
D
atasets
MoFe
A
s
-
isolated
MoFe
O
xidized
MoFe
Alkaline
MoFe
Alkaline
-
inactivated
MoFe
Δ
nif
V
MoFe
Δ
nifV
-
NafT
PDB IDs
8CRS
8DBX
8ENM
8ENL
8ENN
8ENO
Microscope
Titan Krios
Titan Krios
Titan Krios
Titan Krios
Titan Krios
Titan Krios
Camera
Gatan K3
Summit
Gatan K3
Summit
Gatan K3
Summit
Gatan K3
Summit
Gatan K3
Summit
Gatan K3
Summit
Magnification
130,000x
130,000x
130,000x
130,000x
130,000x
130,000x
Voltage (kV)
300
300
300
300
300
300
Recording mode
counting
counting
counting
counting
counting
counting
Frames/Movies
40
40
40
40
40
40
Total Electron dose
(e
-
/Å2)
60
60
60
60
60
60
Defocus range (μm)
-
0.8 to
-
3.0
-
0.8 to
-
3.0
-
0.8 to
-
3.0
-
0.8 to
-
3.0
-
0.8 to
-
3.0
-
0.8 to
-
3.0
Pixel size (Å)
0.65
0.65
0.65
0.65
0.65
0.65
Micrographs
collected
6,777
8,268
12,564
11,663
5,562
16,547
Total extracted
particles
5,724,570
6,367,415
11,018,717
3,658,912
3,738,519
6,724,011
Refined particles
137,629
219,537
331,604
156,311
92,879
99,332
Symmetry imposed
C2
C2
C2
C1
C1
C1
Nominal Map
Resolution (Å)
2.13
1.92
2.14
2.37
2.58
2.71
FSC threshold
0.143
0.143
0.143
0.143
0.143
0.143
masked/unmasked
1.9/2.0
1.8/1.9
1.9/2.0
2.1/2.1
2.2/2.4
2.5/2.6
Refinement
Initial model used
3U7Q
3U7Q
3U7Q
3U7Q
3U7Q
3U7Q
Number of atoms
Protein
17,884
16,986
17,722
16,242
16,749
16,485
Ligand
ICS:2; CLF:
2; HCA:2;
FE:2; 1N7:4
ICS:2; CLF:
2; 1CL:2;
HCA:2; FE:2
ICS:2; CLF:
2; HCA:2;
FE:2;
ICS:2; CLF:
2; HCA:2;
FE:2; 1N7:2
ICS:2;
CLF: 2;
HCA:2;
FE:2;
1N7:2
ICS:2; CLF:
2; HCA:2;
FE:2;
1N7:2
MapCC (mask/box)
0.90/0.77
0.83/0.75
0.90/0.78
0.88/0.74
0.89/0.76
0.89/0.79
Map sharpening B
-
factor
40
30
40
50
40
50
R.m.s. deviations
Bond lengths (Å)
0.003
0.003
0.003
0.003
0.00
4
0.003
Bond angles (º)
0.631
0.566
0.545
0.524
0.5
52
0.537
MolProbity score
1.49
1.58
1.25
1.51
1.
42
1.55
Clashscore (all atom)
9.26
9.2
4.77
5.48
6.
65
6.92
Rotamer outliers (%)
0.99
0.81
0.75
1.53
0.94
1.53
Ramachandran plot
Favored (%)
98.19
97.54
98.14
97.67
97.
75
97.88
Allowed (%)
1.81
1.86
1.86
2.33
2.
25
2.12
Outliers (%)
0.00
0.00
0.00
0.00
0.00
0.00
Supplementary Table
2
:
Summary of notable features in nitrogenase MoFe
-
protein cryoEM
structures presented in this work.
Feature
Dimer
MoFe
As
-
isolated
MoFe
Alkaline
MoFe
Alkaline
-
inactivated
MoFe
Δ
nif
V
MoFe
Δ
nif
V
-
N
af
T
Subunit
Disorder
Ordered
0
0
0
0
α
36
-
α
40
(5)
Disordered
0
0
α
1
-
α
48;
α
354
-
α
360;
α
376
-
α
416;
α
423
-
α
425
(90
residues
)
α
14
-
α
19;
α
25
-
α
26;
α
36
-
α
40;
α
408
-
α
417 (23
residues
)
α
1
-
α
48;
α
376
-
α
383;
α
390
-
α
398;
α
402
-
α
409;
ε
41
-
ε
48 (82
residues
)
HCA loss
Ordered
No
No
Yes
Yes;
citrate
partial
occupancy
Yes;
citrate
partial
occupancy
Disordered
No
No
Yes
Yes
Yes
Phe300
flip
Ordered
No
No
Yes
No
Yes
Disordered
No
No
Yes
Yes
Yes
His274
flip
Ordered
No
No
Yes
No
Yes
Disordered
No
No
Yes
Yes
Yes
His362
flip
Ordered
No
No
No
No
No
Disordered
No
No
Yes
No
Yes
His451
flip
Ordered
No
No
Yes
No
Yes
Disordered
No
No
Yes
Yes
Yes
His442
rearrang
e
ment
Ordered
No
No
No
No
No
Disordered
No
No
Yes
Yes
No
His
-
triad/quartet
Ordered
No
No
No
No
No
Disordered
No
No
Yes
;
quartet
No
Yes
; triad
Trp253
flip
Ordered
No
No
Yes
Yes
Yes
Disordered
No
No
Yes
No
Yes
Gln93
flip
Ordered
No
No
No
No
No
Disordered
No
No
Yes
No
No
Supplementary Figure
1:
Cryo
-
EM
density
of MoFe
-
protein upon exposure to oxygen or
variable
dose. a
, 1.92
Å resolution cryo
-
EM map of MoFe
O
xidized
.
A sample of the MoFe
-
protein alone was transferred from a sealed
anaerobic vial onto a grid within a benchtop Vitrobot (exposed to air) using a gastight syringe and immediately blotted
and plunge frozen (<10 seconds).
b,
To probe the possible photoreductiv
e effects of the electron beam on the
metalloclusters present in the MoFe
-
protein, we subjected the
anaerobically prepared
sample to a dose of 120 e
-
/Å
2
,
panel b represents the resulting map.
c,
Analysis of the
MoFe
oxidized
map surrounding the P
-
cluster revealed bridging
density between Fe6 and Ser β188, but no bridging density between Fe5 and the backbone amide of Cys α88.
Consequently, the aerobically frozen MoFe
-
protein cryo
-
EM structure corresponds to
the oxidized P
+1
st
ate of the P
-
cluster, emphasizing the necessity for anaerobic cryo
-
EM sample preparation conditions for the study of MoFe
-
nitrogenase
d
, Density around the metalloclusters in the low dose (30 e
-
/Å
2
) map.
e,
Density around the metalloclusters
in the high do
se (120 e
-
/Å
2
) map.
Abbreviations: e
-
/Å
2
, electrons per
Å
ngstroms squared; HCA,
R
-
homocitrate.
Supplementary Figure
2
:
Detergent supplementation limits air water interface adsorption and preferred
orientation of MoFe
-
nitrogenase particles
. a,
2.56 Å resolution (C1
-
symmetry) cryo
-
EM map of the MoFe
-
protein
shaded according to local resolution overlaid with surface representation of MoFe
-
protein crystal structure (PDB code
3U7Q).
b,
Cryo
-
EM ESP at 7.5 σ of the metalloclusters overlaid with a rigi
d body fit of PDB code 3U7Q.
c
-
k,
Top
panel:
MoFe
-
protein particles alone on holey carbon grids. Middle panel: MoFe
-
protein particles alone on ultrathin
carbon layered grids. Bottom panel: MoFe
-
protein particles with detergent on holey carbon grids.
(
c, f,
i
) reconstructed
tomogram of MoFe
-
protein particles. Scale bar represents 100 nm (
d, g, j
) Top: Volumetric representation of
tomogram showing particle distribution in ice. The solid gray features correspond to the reconstructed volumes of the
hole edge. B
ottom: Representative 2D classes from corresponding single particle reconstructions. (
e, h, k
) Euler angle
distributions from reconstructed single particle cryo
-
EM maps (Red, overrepresented views).
Abbreviations: Å,
Ångstroms; HCA,
R
-
homocitrate
; nm, nano
meters
.
Supplementary Figure
3
: Anaerobic, as
-
isolated MoFe
-
protein reconstruction with detergent.
a
,
Representative
micrograph of MoFe
-
protein particles on holey carbon grids with CHAPSO
from a dataset of
6,777
micrographs
.
Scale bar corresponds to 50 nm
. b,
Half map FSC curve for reconstructed volume with C2 symmetry imposed. Curve
correlates to FSC calculated with a tight mask.
c,
Cryo
-
EM map color
-
coded according to local resolution estimates.
d,
Cryo
-
EM map color
-
coded according to local resolution estima
tes at high threshold showing local resolution at
metallocluster sites.
e,
Model color
-
coded according to B
-
factor.
f,
Electrostatic potential (
ESP
) map
for bound
CHAPSO molecules.
g,
Cryo
-
EM ESP for
the
previously identified
mononuclear metal binding site
modeled as Fe
with the map sharpened or blurred at indicated B
-
factors.
This site is distinct from other coordination sites identified
within this study
, such as that shown in Figures 3, 4, and
Supplementary Figure
8
.
Abbreviations: Å, Ångstroms;
HCA,
R
-
homocitrate;
nm,
nanometers
;
FSC,
Fourier
Shell
Correlation;
CHAPSO,
3
-
([3
-
Cholamidopropyl]dimethylammonio)
-
2
-
hydroxy
-
1
-
propanesulfonate
.
Supplementary Figure
4
:
MoFe
Alkaline
-
inactivated
exhibits altered biochemical properties
. a,
Separation of
MoFe
A
s
-
isolated
(solid line, yellow),
MoFe
Alkaline
(dashed
-
dotted line, green), and
MoFe
Alkaline
-
inactivated
(dashed line, blue)
by size
ex
cl
usion chromatography monitoring the elution of the
metalloproteins by absorbance at 410 nm
.
b,
Specific activity
assay monitoring inhibition and inactivation of MoFe
-
protein
at pH 9.5
overtime with (+ATP
, green circles
) or without
(
-
ATP
, blue circles
) turnover
(n=2
reactions per condition
, technical replicates)
.
c,
Electron paramagnetic
resonance
spectra
of the
MoFe
Alkaline
-
inactivated
and Fe
-
protein isolated from the alkaline inactivation reactions (Fe
Alkaline
-
inactivated
)
and control proteins
.
Source data are
provided as a Source Data file
.
Abbreviations: ATP, Adenosine triphosphate;
Fe
A
s
-
isolated
, purified Fe
-
protein; Fe
Alkaline
, Fe
-
protein isolated from control, no turnover (
-
ATP) alkaline reactions;
Fe
Alkaline
-
inactivated
, Fe
-
protein isolated from alkaline t
urnover reactions
; C
2
H
4
, ethylene;
mAbs, milli
-
absorbance units
;
mT, milliTesla
.
Supplementary Figure
5
:
Single particle cryoEM characterization of
MoFe
Alkaline
control structure
. a,
Representative micrograph of
MoFe
Alkaline
particles
from a dataset of 12,564 micrographs
.
b,
Representative 2D classes
of
MoFe
Alkaline
particles
c,
H
alf map FSC curve for reconstructed volume
.
d
,
Cryo
-
EM map color
-
coded according to
local resolution estimates.
e
,
Euler angle distributions from reconstructed single particle cryo
-
EM map (Red,
overrepresented views).
f,
Volumetric representation of tomogram showing particle distribution in ice. The so
lid gray
features correspond to the reconstructed volumes of the hole edge.
g
, CryoEM data processing workflow.
Abbreviations: Å, Ångstroms; nm, nanometers; FSC, Fourier Shell Correlation; px, pixel; CTF,
contrast transfer
function.
Supplementary Figure
6
:
Single particle cryoEM characterization of
MoFe
Alkaline
-
inactivated
. a,
Representative
micrograph of
MoFe
Alkaline
-
inactivated
particles
from a dataset of 11,663 micrographs
.
b,
Representative 2D classes of
MoFe
Alkaline
-
inactivated
particles
c,
H
alf map FSC curve for reconstructed volume
.
d
,
Cryo
-
EM map color
-
coded
according to local resolution estimates.
e
,
Euler angle distributions from reconstructed single particle
cryo
-
EM map
(Red, overrepresented views).
f,
Volumetric representation of tomogram showing particle distribution in ice. The solid
gray features correspond to the reconstructed volumes of the hole edge.
g
, CryoEM data processing workflow.
Abbreviations: Å,
Ångstroms; nm, nanometers; FSC, Fourier Shell Correlation; px, pixel; CTF, contrast transfer
function.