EMDB-26648: The CryoEM structure of N49-P9.6-FR3 and PGT121 Fabs in complex w...

Basic information

Entry

Database: EMDB / ID: EMD-26648

• Title: The CryoEM structure of N49-P9.6-FR3 and PGT121 Fabs in complex with BG505 SOSIP.664
• Map data: Phenix sharpened map.

Sample

• Complex: One BG505 SOSIP.644 trimer in complex with three N49-P9.6-FR3 Fabs and three PGT121 Fabs.
  • Protein or peptide: Envelope glycoprotein gp120
  • Protein or peptide: Envelope glycoprotein gp41
  • Protein or peptide: N49-P9.6-FR3 Fab heavy chain
  • Protein or peptide: N49-P9.6-FR3 Fab light chain
  • Protein or peptide: PGT121 Fab light chain
  • Protein or peptide: PGT121 Fab heavy chain
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Keywords: HIV-1 / envelope trimer / GP120 / GP41 / viral entry / type-1 membrane fusion glycoprotein / N49P9.6-FR3 / PGT121 / VIRAL PROTEIN-IMMUNE SYSTEM complex

Function / homology

Function:

positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / apoptotic process / host cell plasma membrane / structural molecule activity / virion membrane / identical protein binding / plasma membrane

Domain/homology:

Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120

Component:

Envelope glycoprotein gp160

• Biological species: Homo sapiens (human) / Human immunodeficiency virus 1
• Method: single particle reconstruction / cryo EM / Resolution: 4.02 Å
• Authors: Nguyen DN / Tolbert WD / Pazgier M

Funding support

United States, 1 items

Organization	Grant number	Country
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	R01AI147870	United States

• Citation: Journal: To Be Published; Title: The structure of N49-P9.6-FR3 and PGT121 Fabs in complex with BG505 SOSIP; Authors: Nguyen DN / Tolbert WD / Pazgier M

History

Deposition	Apr 13, 2022	-
Header (metadata) release	May 3, 2023	-
Map release	May 3, 2023	-
Update	Oct 16, 2024	-
Current status	Oct 16, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_26648.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Phenix sharpened map.
• Voxel size: X=Y=Z: 1.38953 Å

Density

Contour Level	By AUTHOR: 3.8
Minimum - Maximum	-48.407800000000002 - 49.328429999999997
Average (Standard dev.)	0.000000000004753 (±1.0)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Z Y X Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 355.72 Å α=β=γ: 90.0 °

Supplemental data

Half map: Half map B used to generate sharpened map.

• File: emd_26648_half_map_1.map
• Annotation: Half map B used to generate sharpened map.

Half map: Half map A used to generate sharpened map.

• File: emd_26648_half_map_2.map
• Annotation: Half map A used to generate sharpened map.

Sample components

Entire : One BG505 SOSIP.644 trimer in complex with three N49-P9.6-FR3 Fab...

• Entire: Name: One BG505 SOSIP.644 trimer in complex with three N49-P9.6-FR3 Fabs and three PGT121 Fabs.

Components

• Complex: One BG505 SOSIP.644 trimer in complex with three N49-P9.6-FR3 Fabs and three PGT121 Fabs.
  • Protein or peptide: Envelope glycoprotein gp120
  • Protein or peptide: Envelope glycoprotein gp41
  • Protein or peptide: N49-P9.6-FR3 Fab heavy chain
  • Protein or peptide: N49-P9.6-FR3 Fab light chain
  • Protein or peptide: PGT121 Fab light chain
  • Protein or peptide: PGT121 Fab heavy chain
• Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

Supramolecule #1: One BG505 SOSIP.644 trimer in complex with three N49-P9.6-FR3 Fab...

• Supramolecule: Name: One BG505 SOSIP.644 trimer in complex with three N49-P9.6-FR3 Fabs and three PGT121 Fabs.; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6; Details: Fab fragments were generated from papain digest of their respective IgGs. Each component was purified separately. Components were mixed and the complex was purified by gel filtration.
• Source (natural): Organism: Homo sapiens (human)

Macromolecule #1: Envelope glycoprotein gp120

• Macromolecule: Name: Envelope glycoprotein gp120 / type: protein_or_peptide / ID: 1 / Details: Env mimic BG505 SOSIP.664 / Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Human immunodeficiency virus 1
• Molecular weight: Theoretical: 54.064277 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

AENLWVTVYY GVPVWKDAET TLFCASDAKA YETEKHNVWA THACVPTDPN PQEIHLENVT EEFNMWKNNM VEQMHTDIIS LWDQSLKPC VKLTPLCVTL QCTNVTNNIT DDMRGELKNC SFNMTTELRD KKQKVYSLFY RLDVVQINEN QGNRSNNSNK E YRLINCNT SAITQACPKV SFEPIPIHYC APAGFAILKC KDKKFNGTGP CPSVSTVQCT HGIKPVVSTQ LLLNGSLAEE EV MIRSENI TNNAKNILVQ FNTPVQINCT RPNNNTRKSI RIGPGQAFYA TGDIIGDIRQ AHCNVSKATW NETLGKVVKQ LRK HFGNNT IIRFANSSGG DLEVTTHSFN CGGEFFYCNT SGLFNSTWIS NTSVQGSNST GSNDSITLPC RIKQIINMWQ RIGQ AMYAP PIQGVIRCVS NITGLILTRD GGSTNSTTET FRPGGGDMRD NWRSELYKYK VVKIEPLGVA PTRCKRRVVG RRRRR R

UniProtKB: Envelope glycoprotein gp160

Macromolecule #2: Envelope glycoprotein gp41

• Macromolecule: Name: Envelope glycoprotein gp41 / type: protein_or_peptide / ID: 2 / Details: Env mimic BG505 SOSIP.664 / Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Human immunodeficiency virus 1
• Molecular weight: Theoretical: 17.146482 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

AVGIGAVFLG FLGAAGSTMG AASMTLTVQA RNLLSGIVQQ QSNLLRAPEA QQHLLKLTVW GIKQLQARVL AVERYLRDQQ LLGIWGCSG KLICCTNVPW NSSWSNRNLS EIWDNMTWLQ WDKEISNYTQ IIYGLLEESQ NQQEKNEQDL LALD

UniProtKB: Envelope glycoprotein gp160

Macromolecule #3: N49-P9.6-FR3 Fab heavy chain

• Macromolecule: Name: N49-P9.6-FR3 Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.024129 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

HVQLVQSGGG VKKIGAAVRI SCEVSGYNFM DQFINWVRQA PGQGLEWMGW MNPIYGQVNY SWRFQGRVTM TRQLSQDPDD PDWGTAFME LRGLRVDDTA VYYCARGPSG ENYPFHYWGQ GVRVVVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SC

Macromolecule #4: N49-P9.6-FR3 Fab light chain

• Macromolecule: Name: N49-P9.6-FR3 Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 21.733291 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

LTQPASMSAS PGQSVTISCS GTRHIISAWF QQYPGKPPKL IIFDDDKRPS GVPSRFSASR PGDTASLTIS NVQPEDEATY ICNTYEFFG GGTKLTVLGQ PKAAPSVTLF PPSSEELQAN KATLVCLVSD FYPGAVTVAW KADGSPVKVG VETTKPSKQS N NKYAASSY LSLTPEQWKS HRSYSCRVTH EGSTVEKTVA PAECS

Macromolecule #5: PGT121 Fab light chain

• Macromolecule: Name: PGT121 Fab light chain / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 22.869338 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

QVQIDISVAP GETARISCGE KSLGSRAVQW YQHRAGQAPS LIIYNNQDRP SGIPERFSGS PDSPFGTTAT LTITSVEAGD EADYYCHIW DSRVPTKWVF GGGTTLTVLG QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA G VETTTPSK QSNNKYAASS YLSLTPEQWK SHKSYSCQVT HEGSTVEKTV APTEC

Macromolecule #6: PGT121 Fab heavy chain

• Macromolecule: Name: PGT121 Fab heavy chain / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 25.16923 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

QMQLQESGPG LVKPSETLSL TCSVSGASIS DSYWSWIRRS PGKGLEWIGY VHKSGDTNYS PSLKSRVNLS LDTSKNQVSL SLVAATAAD SGKYYCARTL HGRRIYGIVA FNEWFTYFYM DVWGNGTQVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSC

Macromolecule #12: 2-acetamido-2-deoxy-beta-D-glucopyranose

• Macromolecule: Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 12 / Number of copies: 15 / Formula: NAG
• Molecular weight: Theoretical: 221.208 Da

Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.2 / Details: Phosphate buffered saline
• Grid: Model: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
• Details: Quantifoil R1.2/1.3

Electron microscopy

• Microscope: TFS GLACIOS
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4368 / Average electron dose: 58.8 e/Ų
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.5 µm
• Sample stage: Cooling holder cryogen: NITROGEN

Image processing

• Details: Data sets from two grid types were combined into a single data set.
• Particle selection: Number selected: 3151423

Startup model

Type of model: PDB ENTRY
PDB model - PDB ID:

6cdi

Details: 6CDI was used for the BG505 SOSIP.664, 5CEZ was used for the PGT121 Fab, and 7SX7 was used for the N49-P9.6-FR3 Fab.

• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₃ (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX / Number images used: 132055
• Initial angle assignment: Type: OTHER / Software - Name: UCSF ChimeraX
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: PHENIX
• Final 3D classification: Number classes: 1 / Avg.num./class: 132055 / Software - Name: cryoSPARC

Atomic model buiding 1

Initial model

PDB ID	Chain
6cdi	chain_id: G, residue_range: 31-505, source_name: PDB, initial_model_type: experimental model
6cdi	chain_id: A, residue_range: 31-505, source_name: PDB, initial_model_type: experimental model
6cdi	chain_id: I, residue_range: 31-505, source_name: PDB, initial_model_type: experimental model
6cdi	chain_id: B, residue_range: 513-664, source_name: PDB, initial_model_type: experimental model
6cdi	chain_id: C, residue_range: 513-664, source_name: PDB, initial_model_type: experimental model
6cdi	chain_id: J, residue_range: 513-664, source_name: PDB, initial_model_type: experimental model
5cez	chain_id: h, residue_range: 1-112, source_name: PDB, initial_model_type: experimental model
5cez	chain_id: d, residue_range: 1-112, source_name: PDB, initial_model_type: experimental model
5cez	chain_id: k, residue_range: 1-112, source_name: PDB, initial_model_type: experimental model
5cez	chain_id: l, residue_range: 6-107, source_name: PDB, initial_model_type: experimental model
5cez	chain_id: e, residue_range: 6-107, source_name: PDB, initial_model_type: experimental model
5cez	chain_id: m, residue_range: 6-107, source_name: PDB, initial_model_type: experimental model
7sx7	chain_id: H, residue_range: 1-112, source_name: PDB, initial_model_type: experimental model
7sx7	chain_id: D, residue_range: 1-112, source_name: PDB, initial_model_type: experimental model
7sx7	chain_id: K, residue_range: 1-112, source_name: PDB, initial_model_type: experimental model
7sx7	chain_id: L, residue_range: 3-107, source_name: PDB, initial_model_type: experimental model
7sx7	chain_id: E, residue_range: 3-107, source_name: PDB, initial_model_type: experimental model
7sx7	chain_id: M, residue_range: 3-107, source_name: PDB, initial_model_type: experimental model

• Refinement: Space: REAL / Protocol: FLEXIBLE FIT

Output model

PDB-7uoj:
The CryoEM structure of N49-P9.6-FR3 and PGT121 Fabs in complex with BG505 SOSIP.664