A subunit of alpha-1 antitrypsin polymers isolated from ZZ explant liver tissue and decorated with conformationally nonselective Fab 9C5

2025-11-26 REL PDBe PDBe PDBe 2025-01-31 2025-11-26 2025-11-26 2025-11-26 Medical Research Council (MRC, United Kingdom) MR/V034243/1 United Kingdom Other private Alpha-1 Foundation to JAI 1036784 Other government NIHR Investigator awarded to DAL A subunit of alpha-1 antitrypsin polymers isolated from ZZ explant liver tissue and decorated with conformationally nonselective Fab 9C5 Aldobiyan IF Irving JA Orlova EV Lomas DA Alpha-1 antitrypsin, Alpha-1 antitrypsin polymer, Antitrypsin, AAT, Serpin, Serine protease inhibitor, Misfolding, Protein misfolding, Protein oligomer, Oligomerisation, Cirrhosis, Liver tissue, Ex-vivo polymer, Antibody, Antibody-antigen complex, Complex, Fragment antigen-binding region, Fab, PROTEIN BINDING Aldobiyan I Elliston ELK Heyer-Chauhan N Arold ST Zhao L Huntington B Lowen SM Orlova EV Irving JA Lomas DA The mechanism of pathogenic alpha 1 -antitrypsin aggregation in the human liver. Proc.Natl.Acad.Sci.USA US 122 e2507535122 e2507535122 2025 41231946 doi:10.1073/pnas.2507535122 1091-6490 0040 PNASA6 PDB 9HUD unspecified

9C5 Fab in complex with RCL-inserted alpha-1-antitrypsin

PDB 9GJV unspecified

9C5 Fab alone

EMDB EMD-52659 associated EM volume Complex of the 9C5 Fab fragment and a subunit of polymers of the severe deficiency Z alpha-1 antitrypsin variant Complex of the 9C5 Fab fragment and a subunit of polymers of the severe deficiency Z alpha-1 antitrypsin variant 0 1 2 3 Homo sapiens 0.05 Alpha-1 antitrypsin Z variant 1 1

The Z severe deficiency variant of alpha-1 antitrypsin.

Homo sapiens Liver Explant liver tissue ER-derived Inclusion bodies 9C5 Fab 1 2 3 Mus musculus BALB/c Spleen Secreted Alpha-1 antitrypsin Z variant Homo sapiens Liver Explant tissue

The Z severe deficiency variant of alpha-1 antitrypsin E342K variant

LEVO MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDKKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK P01009 9C5 Fab heavy chain Mus musculus BALB/c Spleen

Heavy chain of the Fab fragment of a conformationally nonselective antibody (9C5) that binds to alpha-1-antitrypsin

LEVO QVQLQQSGAELVKPGASVKLSCTATGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATLTADTSSNTAYLQLSSLTSEDTAVYYCARKRYSMDYWGQGTSVTVSSAKTTPPSVYPLAPGSGAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD 9C5 Fab light chain Mus musculus BALB/c Spleen

Light chain of the Fab fragment of a conformationally nonselective antibody (9C5) that binds to alpha-1-antitrypsin

LEVO SIVMTQTPKFLLVSAGERVTITCKASQSVSNDVGWYQQKPGQPPKLLIYNASNRKNGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDHSFPLKFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE singleParticle particle 0.5 7.4 50.0 2-Amino-2-(hydroxymethyl)propane-1,3-diol Tris 50.0 NaCl Sodium chloride 5.0 EDTA

50mM Tris pH 7.4, 50mM NaCl, 5mM EDTA, 0.02 % (w/v) NaN3

C-flat-1.2/1.3 GOLD 400 CARBON HOLEY 20.0 GLOW DISCHARGE 30 AIR ETHANE 94 277 FEI VITROBOT MARK IV

Incubation time: 0s Blot time: 3.5s Blot force: 0s.

The sample comprised a mixed population of unbranched Z alpha-1 antirypsin polymers of varying length (primarily between 4-6 subunits), labelled with the Fab fragment of the 9C5 antibody

0.05 7.4 50.0 2-Amino-2-(hydroxymethyl)propane-1,3-diol Tris 50.0 NaCl Sodium chloride 5.0 EDTA

50mM Tris pH 7.4, 50mM NaCl, 5mM EDTA, 0.02 % (w/v) NaN3

C-flat-1.2/1.3 GOLD 400 CARBON HOLEY 20.0 GLOW DISCHARGE 30 AIR ETHANE 94 277 FEI VITROBOT MARK IV

Incubation time: 0s Blot time: 3.5s Blot force: 0s.

The sample comprised a mixed population of unbranched Z alpha-1 antirypsin polymers of varying length (primarily between 4-6 subunits), labelled with the Fab fragment of the 9C5 antibody

0.05 7.4 50.0 2-Amino-2-(hydroxymethyl)propane-1,3-diol Tris 50.0 NaCl Sodium chloride 5.0 EDTA

50mM Tris pH 7.4, 50mM NaCl, 5mM EDTA, 0.02 % (w/v) NaN3

C-flat-1.2/1.3 GOLD 400 CARBON HOLEY 20.0 GLOW DISCHARGE 30 AIR ETHANE 94 277 FEI VITROBOT MARK IV

Incubation time: 0s Blot time: 3.5s Blot force: 0s.

The sample comprised a mixed population of unbranched Z alpha-1 antirypsin polymers of varying length (primarily between 4-6 subunits), labelled with the Fab fragment of the 9C5 antibody

TFS KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 1.5 2.7 130000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN 88.0 89.0 GIF Bioquantum 20 FEI FALCON IV (4k x 4k) 2 11968 4.0 49.81

Each movie had 1323 raw frames 2 shots per hole Data was collected on a TFS Krios G4 fitted with a (Selectrix X energy filter using a slit width of 20 eV)

TFS KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 1.5 2.7 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN 88.0 89.0 GATAN K3 BIOQUANTUM (6k x 4k) 2 15366 1.15 43.55

Hardware microscope Titan Krios D3771 acceleration voltage keV 300 detector post GIF K3 detector mode super resolution bin 2 Data acquisition parameters nominal magnification 130kx pixel size A 0.65 spot size 4 illuminated area um 0.88 Dose dose per pixel per second e: 18.4 e/px/s dose per A2 per second e: 43.55 e/A2 exposure time s 1.15 number of fractions 50 if Falcon used were the frames aligned no Data collection parameters defocus range from -2.7 defocus range to -1.5 autofocus after centering drift measurement none delay after stage shift s 4. delay after image shift s 0.75 exposures per hole 3 Apertures, size in um c1 2000 c2 50 c3 2000 objective 100 number of images ~15,000 at 3 shots per hole, faster data acquisition, ~640 images per hr Gatan energy filter with a 20 eV slit was used during data collection

TFS KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 1.7 2.7 130000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN 88.0 89.0 TFS Selectris X 20 GATAN K3 BIOQUANTUM (6k x 4k) 1 7982 1.05 38.32

Hardware microscope Titan Krios D3771 acceleration voltage keV 300 detector post GIF K3 detector mode super resolution bin 2 Data acquisition parameters nominal magnification 130kx pixel size A 0.65 spot size 4 illuminated area um 0.92 Dose dose per pixel per second e: 16.2 e/px/s dose per A2 per second e: 38.32 e/A2 exposure time s 1.05 number of fractions 40 if Falcon used were the frames aligned no Data collection parameters defocus range from -2.7 defocus range to -1.5 autofocus after centering drift measurement none delay after stage shift s 4. delay after image shift s 0.75 exposures per hole 3 Apertures, size in um c1 2000 c2 70 c3 2000 objective 100 number of images ~8,000 at 2 shots per hole, faster data acquisition, ~730 images per hr Gatan energy filter with a 20 eV slit was used during data collection

1 5084114 CTFFIND 4.0 CTFFIND 4.1 PHASE FLIPPING AND AMPLITUDE CORRECTION

The initial reference was a lower-resolution cryo-EM map of the Z alpha-1 antitrypsin polymer sample decorated with an alternative Fab fragment (4B12) which had been refined to a nominal 5.7A resolution. The density of antitrypsin remained the same, and the density for the fab fragment was reoriented to correspond with the known epitope of the 9C5 fab - The crystal structure of the complex between antytrpsin and 9C5 Fab has been deposited into the PDB (Accession code 9HUD). During the first round of 3D classification the model was low-pass filtered to 20A. The map was binarise and a large soft-edged mask was used (binary map was extended by 7 pixels, soft-edge of 11 pixels)

FOURIER SPACE 3.98 FSC 0.143 CUT-OFF RELION 5.0-beta-2 273961 MAXIMUM LIKELIHOOD RELION 5.0-beta-2 MAXIMUM LIKELIHOOD RELION 5.0 3 354230.0 RELION 5.0

Class 1: 273961 Class 2: 307137 Class 3: 481591

emd_52659.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 180 180 180 0 0 0 180 180 180 167.4 167.4 167.4 90.0 90.0 90.0 X Y Z -0.00717385 0.020572502 0.00012656288 0.0010935802 0.92999995 0.92999995 0.92999995 0.00516 AUTHOR ::::EMDATABANK.org::::EMD-52659:::: The EM map of the monomer of ex vivo ZZ alpha-1 antitrypsin polymers in complex with a conformationally unselective Fab fragment. 9HUD A 1-354 PDB experimental model

Alpha-1 antitrypsin N-terminal fragment

9HUD B 362-393 PDB experimental model

Alpha-1 antitrypsin C-terminal fragment

9HUD H 1-213 PDB experimental model

9C5 Fab heavy chain

9HUD L 1-213 PDB experimental model

9C5 Fab light chain

RIGID BODY FIT

Rigid body fitting into the EM density was performed in ChimeraX using the backbone trace of the crystal structure of the complex between the RCL-inserted form of alpha-1-antitrypsin and 9C5 Fab

Cross-correlation coefficient REAL emd_52659_msk_1.map emd_52659_additional_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 180 180 180 0 0 0 180 180 180 167.4 167.4 167.4 90.0 90.0 90.0 X Y Z -0.023324115 0.041430876 0.00016029872 0.0014733439 0.92999995 0.92999995 0.92999995 AUTHOR ::::EMDATABANK.org::::EMD-52659:::: The post-processed EM map (masked). B-factor 150. emd_52659_additional_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 180 180 180 0 0 0 180 180 180 167.4 167.4 167.4 90.0 90.0 90.0 X Y Z -0.023324115 0.041430876 0.00012836506 0.0015541451 0.92999995 0.92999995 0.92999995 AUTHOR ::::EMDATABANK.org::::EMD-52659:::: The post-processed EM map. B-factor 150. emd_52659_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 180 180 180 0 0 0 180 180 180 167.4 167.4 167.4 90.0 90.0 90.0 X Y Z -0.007531266 0.021667628 0.00012829318 0.0011332212 0.92999995 0.92999995 0.92999995 AUTHOR ::::EMDATABANK.org::::EMD-52659:::: Half-map 1 of: The EM map of the monomer of ex vivo ZZ alpha-1 antitrypsin polymers in complex with a conformationally unselective Fab fragment. emd_52659_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 180 180 180 0 0 0 180 180 180 167.4 167.4 167.4 90.0 90.0 90.0 X Y Z -0.008137317 0.020783544 0.00012843696 0.001133377 0.92999995 0.92999995 0.92999995 AUTHOR ::::EMDATABANK.org::::EMD-52659:::: Half-map 2 of: The EM map of the monomer of ex vivo ZZ alpha-1 antitrypsin polymers in complex with a conformationally unselective Fab fragment.