i
wwPDB EM Validation Summary Report ○Aug 5, 2024 – 02:24 PM JST
PDB ID : 8WJ1EMDB ID : EMD-37575
Title : cryo-EM structure of human haemoglobin in oxy form
Authors : Takahashi, K.; Lee, Y.; Fago, A.; Bautista, N.M.; Kawamoto, A.; Kurisu, G.;
Storz, J.; Nishizawa, T.; Tame, J.R.H.
Deposited on : 2023-09-25
Resolution : 2.27 Å(reported)
Based on initial model : 2dn1This is a wwPDB EM Validation Summary Report for a publicly released PDB entry.
We welcome your comments at validation@mail.wwpdb.org
A user guide is available athttps://www.wwpdb.org/validation/2017/EMValidationReportHelp
i symbol.
with specific help available everywhere you see the ○The types of validation reports are described at
http://www.wwpdb.org/validation/2017/FAQs#types.

i were used in the production of this report:
The following versions of software and data (see references ○)EMDB validation analysis
Mogul
MolProbity
buster-report
Percentile statistics
MapQ
Ideal geometry (proteins)
Ideal geometry (DNA, RNA)
Validation Pipeline (wwPDB-VP)

:
:
:
:
:
:
:
:
:

0.0.1.dev92
1.8.5 (274361), CSD as541be (2020)
4.02b-467
1.1.7 (2018)20191225.v01 (using entries in the PDB archive December 25th 2019)
1.9.13
Engh & Huber (2001)
Parkinson et al. (1996)
2.37.1

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EMD-37575, 8WJ1

i
Overall quality at a glance ○The following experimental techniques were used to determine the structure:
ELECTRON MICROSCOPY
The reported resolution of this entry is 2.27 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in
the following graphic. The table shows the number of entries on which the scores are based.

MetricClashscore
Ramachandran outliers
Sidechain outliers

Whole archive(#Entries)
158937154571
154315

EM structures(#Entries)
4297
4023
3826

The table below summarises the geometric issues observed across the polymeric chains and their fit
to the map. The red, orange, yellow and green segments of the bar indicate the fraction of residues
that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey
segment represents the fraction of residues that are not modelled. The numeric value for eachfraction is indicated below the corresponding segment, with a dot representing fractions <=5%
The upper red bar (where present) indicates the fraction of residues that have poor fit to the EM
map (all-atom inclusion < 40%). The numeric value is given above the bar.
Mol

Chain

Length

1

A

142

2

B

147

Quality of chain

Page 3

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EMD-37575, 8WJ1

i
Entry composition ○There are 5 unique types of molecules in this entry. The entry contains 2366 atoms, of which 0
are hydrogens and 0 are deuteriums.
In the tables below, the AltConf column contains the number of residues with at least one atom
in alternate conformation and the Trace column contains the number of residues modelled with at
most 2 atoms.
• Molecule 1 is a protein called Hemoglobin subunit alpha.
Mol

Chain

Residues

1

A

141

Atoms
Total CN
O S
1069 685 187 194 3

AltConf

Trace

0

0

AltConf

Trace

0

0

• Molecule 2 is a protein called Hemoglobin subunit beta.
Mol

Chain

Residues

2

B

142

Atoms
Total CN
O S
1084 698 188 195 3• Molecule 3 is PROTOPORPHYRIN IX CONTAINING FE (three-letter code: HEM) (formula: C34 H32 FeN4 O4 ) (labeled as "Ligand of Interest" by depositor).

Mol

Chain

Residues

3

A

1

3

B

1

Total
43
Total
43

Atoms
C Fe
34 1
C Fe
34 1

N
4
N
4

O
4
O
4

AltConf00

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wwPDB EM Validation Summary Report

EMD-37575, 8WJ1

• Molecule 4 is OXYGEN MOLECULE (three-letter code: OXY) (formula: O2 ).

Mol

Chain

Residues

4

A

1

4

B

1

Atoms
Total O2
2
Total O
2
2

AltConf

Atoms
Total O
66
66
Total O
57
57

AltConf00• Molecule 5 is water.
Mol

Chain

Residues

5

A

66

5

B

57

0
0

Page 5

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EMD-37575, 8WJ1i
Residue-property plots ○These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The
first graphic for a chain summarises the proportions of the various outlier classes displayed in thesecond graphic. The second graphic shows the sequence view annotated by issues in geometry andatom inclusion in map density. Residues are color-coded according to the number of geometricquality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2
and red = 3 or more. A red diamond above a residue indicates a poor fit to the EM map forthis residue (all-atom inclusion < 40%). Stretches of 2 or more consecutive residues without any
outlier are shown as a green connector. Residues present in the sample, but not in the model, areshown in grey.
• Molecule 1: Hemoglobin subunit alpha


R141

P114
A115
E116

L101

R92

H89

L80
S81

D74
D75
M76
P77

K56

L48

K40
T41
Y42
F43
P44
H45

L34

L29
E30

K16

MET
V1
L2



Chain A:• Molecule 2: Hemoglobin subunit beta

P124
P125

F118
G119
K120
E121

R104


D94
K95
L96

L91


K82

L88


D79
N80
L81

L75

M55
G56
N57
P58
K59
V60
K61
A62
H63

P51

R40

E26
A27
L28
G29
R30

E22

N19

T12

E7
K8
S9

H2
L3
T4
V133

H143
LYS
TYR
HIS

MET
VAL
V126
Q127



Chain B:

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EMD-37575, 8WJ1i
Experimental information ○Property
EM reconstruction method
Imposed symmetry
Number of particles used
Resolution determination method
CTF correction method
Microscope
Voltage (kV)2Electron dose (e− /Å )Minimum defocus (nm)Maximum defocus (nm)Magnification
Image detector
Maximum map value
Minimum map value
Average map value
Map value standard deviation
Recommended contour level
Map size (Å)
Map dimensions
Map angles (◦ )Pixel spacing (Å)

Value
SINGLE PARTICLE
POINT, C2
550843
FSC 0.143 CUT-OFF
PHASE FLIPPING AND AMPLITUDE
CORRECTIONFEI TITAN KRIOS
300
60.0
400
1200
165000GATAN K3 BIOQUANTUM (6k x 4k)
7.680-5.153-0.0010.107
0.75
261.12, 261.12, 261.12
256, 256, 256
90.0, 90.0, 90.0
1.02, 1.02, 1.02

SourceDepositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
Depositor
wwPDB
wwPDB
wwPDB
Depositor

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EMD-37575, 8WJ1i
Model quality ○5.1

iStandard geometry ○Bond lengths and bond angles in the following residue types are not validated in this section:
HEM, OXYThe Z score for a bond length (or angle) is the number of standard deviations the observed value
is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an
outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (orangles).
Mol

Chain

1
2
All

A
B
All

Bond lengths
RMSZ #|Z| >50.52
0/10970.48
0/11120.50
0/2209Bond angles
RMSZ#|Z| >50.79
1/1491 (0.1%)
0.87
2/1512 (0.1%)
0.83
3/3003 (0.1%)

Chiral center outliers are detected by calculating the chiral volume of a chiral center and verifying if
the center is modelled as a planar moiety or with the opposite hand.A planarity outlier is detectedby checking planarity of atoms in a peptide group, atoms in a mainchain group or atoms of asidechain that are expected to be planar.
Mol
1
2
All

Chain
A
B
All

#Chirality outliers
000#Planarity outliers
12
3There are no bond length outliers.
All (3) bond angle outliers are listed below:
Mol
2
2
1

Chain
B
B
A

Res
30
30
92

Type
ARG
ARG
ARG

Atoms
NE-CZ-NH2
NE-CZ-NH1
NE-CZ-NH1

There are no chirality outliers.
All (3) planarity outliers are listed below:
Mol
1
2
2

Chain
A
B
B

Res
92
104
30

Type
ARG
ARG
ARG

Group
Sidechain
Sidechain
Sidechain

Z
6.88
-6.78
-5.15

Observed(o )123.74116.91117.72

Ideal(o )120.30120.30120.30Page 85.2

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EMD-37575, 8WJ1

i
Too-close contacts ○In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms
and hydrogen atoms in the chain respectively. The H(added) column lists the number of hydrogenatoms added and optimized by MolProbity. The Clashes column lists the number of clashes within
the asymmetric unit, whereas Symm-Clashes lists symmetry-related clashes.
Mol
1
2
3
3
4
4
5
5
All

Chain
A
B
A
B
A
B
A
B
All

Non-H
1069
1084
43
43
2
2
66
57
2366

H(model)
0
0
0
0
0
0
0
0
0

H(added)
1073
1077
30
30
0
0
0
0
2210

Clashes
11
20
2
3
0
0
5
5
35

Symm-Clashes
0
0
0
0
0
0
0
0
0

The all-atom clashscore is defined as the number of clashes found per 1000 atoms (includinghydrogen atoms). The all-atom clashscore for this structure is 8.
The worst 5 of 35 close contacts within the same asymmetric unit are listed below, sorted by theirclash magnitude.
Atom-1Atom-2

2:B:95:LYS:HG3
2:B:3:LEU:HD23
3:B:201:HEM:HBB2
1:A:45:HIS:HE1
3:A:201:HEM:HMB1

2:B:96:LEU:HD13
2:B:7:GLU:HG3
3:B:201:HEM:HHC
5:A:319:HOH:O
3:A:201:HEM:HBB2

Interatomic
distance (Å)
1.61
1.62
1.73
1.80
1.80

Clash
overlap (Å)
0.83
0.81
0.69
0.64
0.63

There are no symmetry-related clashes.

5.3

i
Torsion angles ○

5.3.1

i
Protein backbone ○In the following table, the Percentiles column shows the percent Ramachandran outliers of thechain as a percentile score with respect to all PDB entries followed by that with respect to all EM
entries.
The Analysed column shows the number of residues for which the backbone conformation was
analysed, and the total number of residues.

Page 9wwPDB EM Validation Summary Report

EMD-37575, 8WJ1

Mol

Chain

Analysed

Favoured

AllowedOutliers

Percentiles

1

A

139/142 (98%)

138 (99%)1 (1%)

0

100

100

2

B

140/147 (95%)

137 (98%)

2 (1%)

1 (1%)

22

25

All

All

279/289 (96%)

275 (99%)

3 (1%)

1 (0%)

38

40

All (1) Ramachandran outliers are listed below:
Mol
2

Chain
B

Res
80

Type
ASN

5.3.2

i
Protein sidechains ○In the following table, the Percentiles column shows the percent sidechain outliers of the chain
as a percentile score with respect to all PDB entries followed by that with respect to all EM
entries.
The Analysed column shows the number of residues for which the sidechain conformation was
analysed, and the total number of residues.
Mol

Chain

Analysed

RotamericOutliers

Percentiles

1

A

113/114 (99%)

101 (89%)

12 (11%)

6

7

2

B

114/119 (96%)

101 (89%)

13 (11%)

5

5

All

All

227/233 (97%)

202 (89%)

25 (11%)10

6

5 of 25 residues with a non-rotameric sidechain are listed below:
Mol
2
2
2
2
2

Chain
B
B
B
B
B

Res
9
30
143
28
61

Type
SER
ARG
HIS
LEU
LYS

Sometimes sidechains can be flipped to improve hydrogen bonding and reduce clashes. There areno such sidechains identified.
5.3.3

i
RNA ○

There are no RNA molecules in this entry.

Page 10

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EMD-37575, 8WJ1i
Non-standard residues in protein, DNA, RNA chains ○There are no non-standard protein/DNA/RNA residues in this entry.

5.5iCarbohydrates ○There are no monosaccharides in this entry.

5.6

i
Ligand geometry ○4 ligands are modelled in this entry.
In the following table, the Counts columns list the number of bonds (or angles) for which Mogul
statistics could be retrieved, the number of bonds (or angles) that are observed in the model andthe number of bonds (or angles) that are defined in the Chemical Component Dictionary. The
Link column lists molecule types, if any, to which the group is linked. The Z score for a bondlength (or angle) is the number of standard deviations the observed value is removed from theexpected value. A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection.
RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).

3
3
4,1

Bond lengths
Counts RMSZ #|Z| > 2
1,1,1
0.14
0
1,1,1
0.04
0
41,50,50
1.45
7 (17%)

Bond angles
Counts RMSZ #|Z| > 2
45,82,82
2.03
15 (33%)2,4

41,50,50

45,82,82

Mol

Type

Chain

Res

Link

4
4
3

OXY
OXY
HEM

A
B
A

202
202
201

3

HEM

B

201

1.47

5 (12%)

2.12

16 (35%)In the following table, the Chirals column lists the number of chiral outliers, the number of chiral
centers analysed, the number of these observed in the model and the number defined in theChemical Component Dictionary. Similar counts are reported in the Torsion and Rings columns.
’-’ means no outliers of that kind were identified.
Mol
3
3

Type
HEM
HEM

Chain
A
B

Res
201
201

Link
4,1
2,4

Chirals-

Torsions
0/12/54/54
5/12/54/54

Rings
-

The worst 5 of 12 bond length outliers are listed below:
Mol
3
3
3
3

Chain
B
A
A
B

Res
201
201
201
201

Type
HEM
HEM
HEM
HEM

Atoms
CAA-C2AC1B-NB
C1D-ND
FE-NB

Z
-4.79
-3.65
-3.28
3.14

Observed(Å) Ideal(Å)
1.45
1.52
1.34
1.40
1.32
1.38
2.12
1.96
Continued on next page...

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wwPDB EM Validation Summary Report

Continued from previous page...
Mol Chain Res Type
Atoms
3
A
201 HEM
FE-NB

Z
3.14

Observed(Å)
2.12

EMD-37575, 8WJ1

Ideal(Å)
1.96

The worst 5 of 31 bond angle outliers are listed below:
Mol
3
3
3
3
3

Chain
A
A
B
B
B

Res
201
201
201
201
201

Type
HEM
HEM
HEM
HEM
HEM

Atoms
C1B-NB-C4B
CHC-C4B-NB
CBD-CAD-C3D
CHD-C1D-ND
CMB-C2B-C1B

Z
5.04
4.68
-4.20
4.01
3.97

Observed(o )110.28129.51100.95128.79131.09Ideal(o )105.07124.43112.63124.43125.04

There are no chirality outliers.
All (5) torsion outliers are listed below:
Mol
3
3
3
3
3

Chain
B
B
B
B
B

Res
201
201
201
201
201

Type
HEM
HEM
HEM
HEM
HEM

Atoms
C4B-C3B-CAB-CBB
CAD-CBD-CGD-O1D
CAD-CBD-CGD-O2D
CAA-CBA-CGA-O2A
CAA-CBA-CGA-O1A

There are no ring outliers.
2 monomers are involved in 5 short contacts:
Mol
3
3

Chain
A
B

Res
201
201

Type
HEM
HEM

Clashes2
3

Symm-Clashes
00

The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths,
bond angles, torsion angles, and ring geometry for all instances of the Ligand of Interest. In
addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will
also be included. For torsion angles, if less then 5% of the Mogul distribution of torsion angles iswithin 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier.
Any bond that is central to one or more torsion angles identified as an outlier by Mogul will behighlighted in the graph. For rings, the root-mean-square deviation (RMSD) between the ringin question and similar rings identified by Mogul is calculated over all ring torsion angles. If theaverage RMSD is greater than 60 degrees and the minimal RMSD between the ring in question andany Mogul-identified rings is also greater than 60 degrees, then that ring is considered an outlier.
The outliers are highlighted in purple. The color gray indicates Mogul did not find sufficientequivalents in the CSD to analyse the geometry.

Page 12wwPDB EM Validation Summary Report
Ligand HEM A 201

Bond lengths

Bond angles

Torsions

Rings

EMD-37575, 8WJ1

Page 13

wwPDB EM Validation Summary Report
Ligand HEM B 201

5.7

Bond lengths

Bond angles

Torsions

Rings

i
Other polymers ○There are no such residues in this entry.

5.8i
Polymer linkage issues ○There are no chain breaks in this entry.

EMD-37575, 8WJ1

Page 14

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EMD-37575, 8WJ1i
Map visualisation ○This section contains visualisations of the EMDB entry EMD-37575. These allow visual inspectionof the internal detail of the map and identification of artifacts.
Images derived from a raw map, generated by summing the deposited half-maps, are presented
below the corresponding image components of the primary map to allow further visual inspection
and comparison with those of the primary map.

6.1

iOrthogonal projections ○6.1.1

Primary map

X

6.1.2

Y

Z

Y

Z

Raw mapX

The images above show the map projected in three orthogonal directions.

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wwPDB EM Validation Summary Report

6.2

iCentral slices ○6.2.1

Primary map

X Index: 128

6.2.2

EMD-37575, 8WJ1Y Index: 128

Z Index: 128

Y Index: 128

Z Index: 128

Raw map

X Index: 128

The images above show central slices of the map in three orthogonal directions.

Page 16

wwPDB EM Validation Summary Report

6.3

iLargest variance slices ○6.3.1

Primary map

X Index: 134

6.3.2

EMD-37575, 8WJ1Y Index: 111

Z Index: 124

Y Index: 112

Z Index: 121

Raw map

X Index: 133

The images above show the largest variance slices of the map in three orthogonal directions.

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EMD-37575, 8WJ1

6.4

i
Orthogonal standard-deviation projections (False-color) ○6.4.1

Primary map

X

6.4.2

Y

Z

Y

Z

Raw mapX

The images above show the map standard deviation projections with false color in three orthogonal
directions. Minimum values are shown in green, max in blue, and dark to light orange shades
represent small to large values respectively.

Page 18

wwPDB EM Validation Summary Report

6.5

iOrthogonal surface views ○6.5.1

Primary map

XY

EMD-37575, 8WJ1

Z

The images above show the 3D surface view of the map at the recommended contour level 0.75.
These images, in conjunction with the slice images, may facilitate assessment of whether an appropriate contour level has been provided.
6.5.2

Raw map

X

Y

Z

These images show the 3D surface of the raw map. The raw map’s contour level was selected sothat its surface encloses the same volume as the primary map does at its recommended contour
level.

Page 19

6.6

wwPDB EM Validation Summary Report

EMD-37575, 8WJ1

i
Mask visualisation ○This section shows the 3D surface view of the primary map at 50% transparency overlaid with thespecified mask at 0% transparency
A mask typically either:• Encompasses the whole structure• Separates out a domain, a functional unit, a monomer or an area of interest from a larger
structure
6.6.1

i
emd_37575_msk_1.map ○

X

Y

Z

Page 20

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EMD-37575, 8WJ1i
Map analysis ○This section contains the results of statistical analysis of the map.

7.1

i
Map-value distribution ○The map-value distribution is plotted in 128 intervals along the x-axis. The y-axis is logarithmic.
A spike in this graph at zero usually indicates that the volume has been masked.

Page 21

7.2

wwPDB EM Validation Summary Report

EMD-37575, 8WJ1i
Volume estimate ○The volume at the recommended contour level is 25 nm3 ; this corresponds to an approximate massof 23 kDa.
The volume estimate graph shows how the enclosed volume varies with the contour level. The
recommended contour level is shown as a vertical line and the intersection between the line andthe curve gives the volume of the enclosed surface at the given level.

Page 22

7.3

wwPDB EM Validation Summary ReportiRotationally averaged power spectrum ○*Reported resolution corresponds to spatial frequency of 0.441 Å−1

EMD-37575, 8WJ1

Page 23

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EMD-37575, 8WJ1i
Fourier-Shell correlation ○Fourier-Shell Correlation (FSC) is the most commonly used method to estimate the resolution of
single-particle and subtomogram-averaged maps. The shape of the curve depends on the imposed
symmetry, mask and whether or not the two 3D reconstructions used were processed from acommon reference. The reported resolution is shown as a black line. A curve is displayed for thehalf-bit criterion in addition to lines showing the 0.143 gold standard cut-off and 0.5 cut-off.

8.1

i
FSC ○

*Reported resolution corresponds to spatial frequency of 0.441 Å−1Page 24

8.2

wwPDB EM Validation Summary Report

EMD-37575, 8WJ1i
Resolution estimates ○Resolution estimate (Å)
Reported by author
Author-provided FSC curve
Unmasked-calculated*

Estimation criterion (FSC cut-off )0.143 0.5
Half-bit
2.27
2.27 2.58
2.30
2.65 2.85
2.68*Resolution estimate based on FSC curve calculated by comparison of deposited half-maps. The
value from deposited half-maps intersecting FSC 0.143 CUT-OFF 2.65 differs from the reported
value 2.27 by more than 10 %Page 25

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EMD-37575, 8WJ1i
Map-model fit ○This section contains information regarding the fit between EMDB map EMD-37575 and PDB
model 8WJ1. Per-residue inclusion information can be found in section 3 on page 5.

9.1

Map-model overlays

9.1.1

i
Map-model overlay ○

X

9.1.2

Y

Z

Y

Z

i
Map-model assembly overlay ○X

The images above show the 3D surface view of the map at the recommended contour level 0.75 at
50% transparency in yellow overlaid with a ribbon representation of the model coloured in blue.
These images allow for the visual assessment of the quality of fit between the atomic model andthe map.

Page 26

9.2

wwPDB EM Validation Summary Report

EMD-37575, 8WJ1

i
Q-score mapped to coordinate model ○

X

Y

Z

The images above show the model with each residue coloured according its Q-score. This showstheir resolvability in the map with higher Q-score values reflecting better resolvability. Please
note: Q-score is calculating the resolvability of atoms, and thus high values are only expected at
resolutions at which atoms can be resolved. Low Q-score values may therefore be expected formany entries.

9.3

iAtom inclusion mapped to coordinate model ○

X

Y

Z

The images above show the model with each residue coloured according to its atom inclusion. Thisshows to what extent they are inside the map at the recommended contour level (0.75).

Page 27

9.4

wwPDB EM Validation Summary Report

EMD-37575, 8WJ1i
Atom inclusion ○At the recommended contour level, 94% of all backbone atoms, 92% of all non-hydrogen atoms,
are inside the map.

Page 28

9.5

wwPDB EM Validation Summary Report

EMD-37575, 8WJ1i
Map-model fit summary ○The table lists the average atom inclusion at the recommended contour level (0.75) and Q-score
for the entire model and for each chain.

Chain
All
A
B

Atom inclusion
0.92200.94400.9030Q-score0.69200.69500.6900