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- EMDB-43983: The map of the bent AlphaIIbBeta3 in complex with R21D10 Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-43983
TitleThe map of the bent AlphaIIbBeta3 in complex with R21D10 Fab
Map dataIt is the main map.
Sample
  • Complex: The semi-extended AlphaIIbBeta3 in complex with R21D10 Fab
    • Complex: AlphaIIbBeta3 Integrin
      • Protein or peptide: Integrin AlphaIIb
      • Protein or peptide: Integrin Beta3
    • Complex: R21D10 Fab
      • Protein or peptide: R21D10 Fab heavy chain
      • Protein or peptide: R211010 Fab light chain
KeywordsMurine monoclonal antibody R21D10 stabilizes a semi-extended conformation of the platelet AlphaIIbBeta3 integrin receptor and serves as an allosteric inhibitor of ligand binding and platelet aggregation. It also is the first mAb reported to inhibit the binding of protein disulfide isomerase. / BLOOD CLOTTING
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsWang JL / Walz T / Coller B / Wang L / Li JH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)5R01HL019278-48 United States
CitationJournal: Blood Adv / Year: 2024
Title: An αIIbβ3 monoclonal antibody traps a semiextended conformation and allosterically inhibits large ligand binding.
Authors: Lu Wang / Jialing Wang / Jihong Li / Thomas Walz / Barry S Coller /
Abstract: Monoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation ...Monoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation and binds to thrombin-activated αIIbβ3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor-activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate-induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-β3 β-I domain), and immunoblot studies indicated that R21D10 binds to β3. The dissociation of αIIbβ3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbβ3-R21D10 Fab complex revealed that R21D10 binds to the β3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbβ3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the β3 I-EGF2 domain associated with a new interaction between the β3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the β3 hybrid domain required for high-affinity ligand binding and protect αIIbβ3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbβ3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.
History
DepositionMar 7, 2024-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43983.png

Downloads & links

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Map

FileDownload / File: emd_43983.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIt is the main map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 400 pix.
= 344. Å		0.86 Å/pix.
x 400 pix.
= 344. Å		0.86 Å/pix.
x 400 pix.
= 344. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.0794004 - 1.7719426
Average (Standard dev.)-0.000020222735 (±0.037487343)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 344.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: It is the half B map.

Fileemd_43983_half_map_1.map
AnnotationIt is the half B map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: It is the half A map.

Fileemd_43983_half_map_2.map
AnnotationIt is the half A map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The semi-extended AlphaIIbBeta3 in complex with R21D10 Fab

EntireName: The semi-extended AlphaIIbBeta3 in complex with R21D10 Fab
Components
  • Complex: The semi-extended AlphaIIbBeta3 in complex with R21D10 Fab
    • Complex: AlphaIIbBeta3 Integrin
      • Protein or peptide: Integrin AlphaIIb
      • Protein or peptide: Integrin Beta3
    • Complex: R21D10 Fab
      • Protein or peptide: R21D10 Fab heavy chain
      • Protein or peptide: R211010 Fab light chain

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Supramolecule #1: The semi-extended AlphaIIbBeta3 in complex with R21D10 Fab

SupramoleculeName: The semi-extended AlphaIIbBeta3 in complex with R21D10 Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 250 KDa

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Supramolecule #2: AlphaIIbBeta3 Integrin

SupramoleculeName: AlphaIIbBeta3 Integrin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: R21D10 Fab

SupramoleculeName: R21D10 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Integrin AlphaIIb

MacromoleculeName: Integrin AlphaIIb / type: protein_or_peptide / ID: 1
Details: The full-length Integrin AlphaIIb includes a signal peptide.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MARALCPLQA LWLLEWVLLL LGPCAAPPAW ALNLDPVQLT FYAGPNGSQF GFSLDFHKDS HGRVAIVVGA PRTLGPSQEE TGGVFLCPW RAEGGQCPSL LFDLRDETRN VGSQTLQTFK ARQGLGASVV SWSDVIVACA PWQHWNVLEK TEEAEKTPVG S CFLAQPES ...String:
MARALCPLQA LWLLEWVLLL LGPCAAPPAW ALNLDPVQLT FYAGPNGSQF GFSLDFHKDS HGRVAIVVGA PRTLGPSQEE TGGVFLCPW RAEGGQCPSL LFDLRDETRN VGSQTLQTFK ARQGLGASVV SWSDVIVACA PWQHWNVLEK TEEAEKTPVG S CFLAQPES GRRAEYSPCR GNTLSRIYVE NDFSWDKRYC EAGFSSVVTQ AGELVLGAPG GYYFLGLLAQ APVADIFSSY RP GILLWHV SSQSLSFDSS NPEYFDGYWG YSVAVGEFDG DLNTTEYVVG APTWSWTLGA VEILDSYYQR LHRLRGEQMA SYF GHSVAV TDVNGDGRHD LLVGAPLYME SRADRKLAEV GRVYLFLQPR GPHALGAPSL LLTGTQLYGR FGSAIAPLGD LDRD GYNDI AVAAPYGGPS GRGQVLVFLG QSEGLRSRPS QVLDSPFPTG SAFGFSLRGA VDIDDNGYPD LIVGAYGANQ VAVYR AQPV VKASVQLLVQ DSLNPAVKSC VLPQTKTPVS CFNIQMCVGA TGHNIPQKLS LNAELQLDRQ KPRQGRRVLL LGSQQA GTT LNLDLGGKHS PICHTTMAFL RDEADFRDKL SPIVLSLNVS LPPTEAGMAP AVVLHGDTHV QEQTRIVLDC GEDDVCV PQ LQLTASVTGS PLLVGADNVL ELQMDAANEG EGAYEAELAV HLPQGAHYMR ALSNVEGFER LICNQKKENE TRVVLCEL G NPMKKNAQIG IAMLVSVGNL EEAGESVSFQ LQIRSKNSQN PNSKIVLLDV PVRAEAQVEL RGNSFPASLV VAAEEGERE QNSLDSWGPK VEHTYELHNN GPGTVNGLHL SIHLPGQSQP SDLLYILDIQ PQGGLQCFPQ PPVNPLKVDW GLPIPSPSPI HPAHHKRDR RQIFLPEPEQ PSRLQDPVLV SCDSAPCTVV QCDLQEMARG QRAMVTVLAF LWLPSLYQRP LDQFVLQSHA W FNVSSLPY AVPPLSLPRG EAQVWTQLLR ALEERAIPIW WVLVGVLGGL LLLTILVLAM WKVGFFKRNR PPLEEDDEEG E

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Macromolecule #2: Integrin Beta3

MacromoleculeName: Integrin Beta3 / type: protein_or_peptide / ID: 2
Details: The sequence is for the full-length Beta3 including a signal peptide.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MRARPRPRPL WATVLALGAL AGVGVGGPNI CTTRGVSSCQ QCLAVSPMCA WCSDEALPLG SPRCDLKENL LKDNCAPESI EFPVSEARV LEDRPLSDKG SGDSSQVTQV SPQRIALRLR PDDSKNFSIQ VRQVEDYPVD IYYLMDLSYS MKDDLWSIQN L

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Macromolecule #3: R21D10 Fab heavy chain

MacromoleculeName: R21D10 Fab heavy chain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString:
EVQLQESGAE LAKPGASVKM SCKASGYTFT DYWMHWVKQR PGQGLEWIGY INPSTGYTEY NQKFKDKATL TADKSSSTAY MQLSSLTSE DSAVYYCARW RGVYRSDVHY YAMDYWGQGT SVTVSSAKTT APSVYPLAPV CGDTTGSSVT LGCLVKGYFP E

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Macromolecule #4: R211010 Fab light chain

MacromoleculeName: R211010 Fab light chain / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString: QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMHWYQQKSG TSPKRWIYDT SKLASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQ WSSKPPTFGA GTKLELKWAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK ...String:
QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMHWYQQKSG TSPKRWIYDT SKLASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQ WSSKPPTFGA GTKLELKWAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK DSTYSMSSTL TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
DetailsAlphaIIbBeta3 in complex with R21D10 Fab

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 993650
Startup modelType of model: OTHER
Details: generate initial model based on selected averages of particles after 2D-classification in CryoSPARC.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4.0) / Number images used: 107419
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.4.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 183000 / Software - Name: cryoSPARC (ver. v4.4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

8t2v

chain_id: A, source_name: PDB, initial_model_type: experimental model

8t2v

chain_id: B, source_name: PDB, initial_model_type: experimental model

9axl

chain_id: H, source_name: PDB, initial_model_type: experimental model

9axl

chain_id: L, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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