Table 1 Crystallographic data for the 3H4 Fab and VL9-bound HLA-E co-complex structure.

From: Mouse and human antibodies bind HLA-E-leader peptide complexes and enhance NK cell cytotoxicity

a

Data collection

3H4 Fab-HLA-E (VL9)

d

VL9 peptide-Fab HC interface

f

HLA-E HC-Fab LC interface

 

Crystallization

20% PEG 8000

 

Peptide

 

Fab HC

 

HLA-E HC

 

Fab LC

 

Condition

0.1 M NA HEPES

 

P1 Val

 

P97 Tyr (CDR3)

 

P55 Glu (α1)

 

P1 Asp

  

pH 7

 

[CB, CG1, CG2]

 

[CZ, OH]

 

[OE1]

 

[OD1, OD2]

 

Resolution Å

54.74–1.80

 

P2 Met

 

P100 Ser (CDR3)

 

P58 Glu (α1)

 

P27 Gln (CDR1)

  

(1.864– 1.80)

 

[O]

 

[OG]

 

[C, O, CB, CG, CD, OE1, OE2]

 

[NE2]

 

Space group

C2

 

P4 Pro

 

P100A Ser (CDR3)

 

P59 Tyr (α1)

 

P28 Asp (CDR1)

 

Cell dimensions

a = 190.47

 

[C, O, CB, CG, CD]

 

[N, C, O, CB, OG]

 

[N, CA, CB]

 

[O]

  

b = 73.69

 

P5 Arg

 

P100B Tyr (CDR3)

 

P62 Arg (α1)

 

P30 Asn (CDR1)

  

c = 159.75

 

[CB, CG, CD, NH1]

 

[N, CA, CB, CG, CD1, CD2, CE1, CE2, CZ, OH]

 

[CB, CG, CD, NE]

 

[CB, CG, ND2, OD1]

  

α = 90.0°

     

P162 Asp (α2)

 

P32 Tyr (CDR1)

  

β = 101.62°

e

HLA-E HC-Fab HC interface

 

[C, CB, CG, OD2]

 

[CE2, OH]

  

γ = 90.0°

 

HLA-E HC

 

Fab HC

 

P163 Thr (α2)

 

P92 Asp (CDR3)

 

Solvent content [%]

55 (2)

 

P56 Gly (α1)

 

P33 Asn (CDR1)

 

[CA, OG1]

 

[O, CG, OD1, OD2]

 

(copies in AU)

  

[CA]

 

[ND2]

 

P166 Glu (α2)

 

P93 Glu (CDR3)

 

Unique reflections

196,943 (19,307)

 

P57 Ser (α1)

 

P47 Trp

 

[CA, CB, CG, CD, OE1, OE2]

 

[CA, CB, CG, CD, OE1, OE2]

 

Completeness [%]

98.1 (96.8)

 

[N, CB, OG]

 

[CZ3, CH2]

 

P167 Trp (α2)

 

P94 Phe (CDR3)

 

Rmerge [I]

0.057 (1.289)

 

P58 Glu (α1)

 

P50 Asp

 

[NE1, CZ2, CZ3, CH2]

 

[C, O, CB, CG, CD1, CD2, CE1, CE2, CZ]

 

I/sigma

15.0 (0.5)

 

[CA, O, CB, CG, CD, OE1, OE2]

 

[OD1]

 

P170 Lys (α2)

 

P95 Pro (CDR3)

 

Multiplicity

6.9 (7.1)

 

P59 Tyr (α1)

 

P52 Asn (CDR2)

 

[CD, CE, NZ]

 

[N, CA, CB, CG, CD]

 

CC1/2

0.998 (0.724)

 

[CA, O, CB, CD2, CE2]

 

[CB, ND2]

 

P174 Lys (α2)

  
 

Refinement

  

P61 Asp (α1)

 

P54 Asn (CDR2)

 

[CE, NZ]

  
 

No. of non-hydrogen atoms

14,072

 

[C, O, CB, CG, OD1, OD2]

 

[ND2]

    
 

Rfactor [%)]

0.1954 (0.4164)

 

P62 Arg (α1)

 

P56 Gly (CDR2)

g

Hydrogen bonds

 

Rfree [%]

0.2323 (0.4304)

 

[N, CA, CB, CG, CD, NE, CZ, NH2]

 

[CA, C, O]

No.

HLA-E HC

Distance (Å)

Fab HC

 

r.m.s.d. bonds [Å]a

0.005

 

P63 Glu (α1)

 

P57 Thr (CDR2)

1

P62 Arg [NE] (α1)

2.97

P100B Tyr [O] (CDR3)

 

r.m.s.d. angles [deg]

0.69

 

[OE1]

 

[N, CA, C, O]

2

P62 Arg  [NH2] (α1)

2.93

P100A Ser [O]   (CDR3)

 

Ramachandran statistics

  

P65 Arg (α1)

 

P58 Ile

3

P62 Arg  [NH2] (α1)

3.06

P100B Tyr  [O] (CDR3)

 

Favored [%]

98.31

 

[CB, CD, NE, CZ, NH1, NH2]

 

[CA, CG1, CG2, CD1]

4

P65 Arg [NH1] (α1)

2.74

P50 Asp  [OD1]

 

Disallowed [%]

0

 

P66 Ser (α1)

 

P59 Tyr

5

P65 Arg [NH2] (α1)

3.47

P50 Asp [O] 

    

[OG]

 

[N, O]

6

P65 Arg  [NH2] (α1)

2.77

P50 Asp  [OD1]

b

Total buried surface area of the interface (Å2)

P69 Asp (α1)

 

P60 Asn

 

7

P65 Arg  [NH2] (α1)

3.11

P56 Gly  [O]  (CDR2)

 

Peptide-Fab HC

242.6

 

[CA, CG, OD1, OD2]

 

[CA, CG, OD1]

8

P163 Thr  [OG1] (α2)

2.45

P100 Ser [OG] (CDR3)

 

(Chains P-G)

  

P154 Glu (α2)

 

P61 Gln

9

P69 Asp  [OD1] (α1)

3.37

P54 Asn [ND2] (CDR2)

 

HLA-E HC-Fab HC

1109.4

 

[OE1]

 

[N, CB, CD, NE2, OE1]

10

P58 Glu  [OE2] (α1)

2.86

P61 Gln  [N]

 

(Chains A–G)

  

P155 His (α2)

 

P64 Lys

11

P61 Asp  [OD2] (α1)

2.76

P64 Lys [NZ]

 

HLA-E HC-Fab LC

522.8

 

[CA, O, ND1, CE1]

 

[CE, NZ]

 

Salt bridges

 

(Chains A–H)

  

P158 Ala (α2)

 

P97 Tyr (CDR3)

No.

HLA-E HC

Distance (Å)

Fab HC

    

[C, O, CB]

 

[CE2, CZ, OH]

1

P65 Arg [NH1] (α1)

2.74

P50 Asp  [OD1]

c

3H4 Fab-HLA-E (VL9)

RMSD (Å)

 

P159 Tyr (α2)

 

P99 Gly (CDR3)

2

P65 Arg  [NH2] (α1)

2.77

P50 Asp  [OD1]

 

Chains A & C

0.95

 

[N, CA, CB, CD1]

 

[CA, C, O]

3

P61 Asp  [OD2] (α1)

2.76

P64 Lys  [NZ]

 

(HLA-E heavy chain)

  

P162 Asp (α2)

 

P100 Ser (CDR3)

    
 

Chains A & C

0.51

 

[CB, OD2]

 

[N, CA, CB, OG]

h

Hydrogen bonds

 

(HLA-E heavy chain—no α3 domain)

  

P163 Thr (α2)

 

P100A Ser (CDR3)

No.

HLA-E HC

Distance (Å)

Fab LC

 

Chains B & D

0.82

 

[CB, CG2, OG1]

 

[C, O, CB, OG]

1

P170 Lys  [NZ] (α2)

2.80

P92 Asp  [OD2] (CDR3)

 

(β2-microglobulin)

  

P167 Trp (α2)

 

P100B Tyr (CDR3)

2

P170 Lys  [NZ] (α2)

2.69

P93 Glu  [OE2] (CDR3)

 

Chains E & G

0.89

 

[CE2, NE1, CZ2, CH2]

 

[CA, O, CB, CG, CD1, CD2, CE1, CE2, CZ, OH]

3

P166 Glu  [OE2] (α2)

2.89

P30 Asn  [ND2] (CDR1)

 

(Fab heavy chain)

    

P100D Trp (CDR3)

 

Salt bridges

 

Chains F & H

0.36

   

[CZ3, CH2]

No.

HLA-E HC

Distance (Å)

Fab LC

 

(Fab light chain)

     

1

P170 Lys  [NZ]  (α2)

3.96

P92 Asp  [OD1] (CDR3)

 

3H4 Fab-HLA-E(VL9) and 1MHE from O’Callaghan et al. (1998)

RMSD

    

2

P170 Lys  [NZ] (α2)

2.80

P92 Asp  [OD2] (CDR3)

 

Chains A & A

0.669

    

3

P170 Lys  [NZ] (α2)

2.69

P93 Glu  [OE2] (CDR3)

  1. a Crystallographic data collection and refinement statistics. AS ammonium sulfate. ar.m.s.d.: root mean-square deviation from ideal geometry. Statistics for outer shell indicated in parentheses. AU asymmetric unit. Rfree equals the R-factor against 5% of the data removed prior to refinement.
  2. b–f Inter-chain RMSD and inter-molecular interfaces in the 3H4-HLA-E-VL9 structure. b Table detailing the total buried surface area of the interface in Å2 between the 3H4 VH, VL, and the VL9-bound HLA-E complex. c Table of RMSD (root mean-square deviation) in Å between chains of the 3H4-HLA-E-VL9 co-complex structure. Two copies of the 3H4 Fab-HLA-E-VL9 co-complex were present in the asymmetric unit and thus RMSD between chains related by non-crystallographic symmetry was calculated via Cα atom pairwise alignment on the PDBePISA server. Average Cα atom RMSD following pairwise alignment is also reported for the HLA-E heavy chain (HC) of 1MHE (Chain A), a previously published non-receptor-bound VL9-loaded HLA-E complex, and the HLA-E HC from the 3H4-HLA-E-VL9 structure reported here (Chain A). d Table listing residues involved in the interface between the 3H4 VH and the VL9 peptide. e Table of interacting residues of the 3H4 VH and HLA-E HC interface. f Table of interacting residues of the 3H4 VL and HLA-E HC interface.
  3. g, h Hydrogen bonding and salt bridges in the 3H4-HLA-E-VL9 structure. Table of hydrogen bonds and salt bridges formed between the 3H4 heavy chain (HC) and the HLA-E HC (g) and the 3H4 light chain and the HLA-E HC (h). Hydrogen bonding cut-offs according to the PDBePISA default criteria. 3H4 chain numbering is according to the Kabat scheme whereby alternate insertion codes (letters after the residue number) are added to variable length regions of the antibody sequence. 3H4 residues within the CDRs are shaded green and labeled “CDR1/2/3”. The position of HLA-E HC residues either on the α1 or α2 helix is also noted. Amino acid atom abbreviations: C—mainchain Carbon atom, O—mainchain Oxygen atom, N—mainchain Nitrogen atom, CA—α-Carbon atom, CB—β-Carbon atom, CD—δ-Carbon atom, CE—ε-Carbon atom, CG— γ-Carbon atom, CH—η-Carbon atom, CZ—ζ-Carbon atom, OD—δ-Oxygen atom, OE— ε-Oxygen atom, OG—γ-Oxygen atom, OH—η-Oxygen atom, ND—δ-Nitrogen atom, NE—ε-Nitrogen atom, NH—η-Nitrogen atom, NZ—ζ-Nitrogen atom.
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