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- PDB-9hac: De novo designed BBF-14 beta barrel with computationally designed... -

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Basic information

Entry
Database: PDB / ID: 9hac
TitleDe novo designed BBF-14 beta barrel with computationally designed BBF-14_b4 binder
Components
  • BBF-14
  • BBF-14_binder4
KeywordsDE NOVO PROTEIN / beta barrel / BBF-14 / computational / binder
Function / homology:
Function and homology information
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsPacesa, M. / Nickel, L. / Correia, B.E.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: bioRxiv / Year: 2024
Title: BindCraft: one-shot design of functional protein binders.
Authors: Martin Pacesa / Lennart Nickel / Christian Schellhaas / Joseph Schmidt / Ekaterina Pyatova / Lucas Kissling / Patrick Barendse / Jagrity Choudhury / Srajan Kapoor / Ana Alcaraz-Serna / ...Authors: Martin Pacesa / Lennart Nickel / Christian Schellhaas / Joseph Schmidt / Ekaterina Pyatova / Lucas Kissling / Patrick Barendse / Jagrity Choudhury / Srajan Kapoor / Ana Alcaraz-Serna / Yehlin Cho / Kourosh H Ghamary / Laura Vinué / Brahm J Yachnin / Andrew M Wollacott / Stephen Buckley / Adrie H Westphal / Simon Lindhoud / Sandrine Georgeon / Casper A Goverde / Georgios N Hatzopoulos / Pierre Gönczy / Yannick D Muller / Gerald Schwank / Daan C Swarts / Alex J Vecchio / Bernard L Schneider / Sergey Ovchinnikov / Bruno E Correia
Abstract: Protein-protein interactions (PPIs) are at the core of all key biological processes. However, the complexity of the structural features that determine PPIs makes their design challenging. We present ...Protein-protein interactions (PPIs) are at the core of all key biological processes. However, the complexity of the structural features that determine PPIs makes their design challenging. We present BindCraft, an open-source and automated pipeline for protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, designed proteins, and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity, and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Lastly, we utilize cell surface receptor-specific binders to redirect AAV capsids for targeted gene delivery. This work represents a significant advancement towards a "one design-one binder" approach in computational design, with immense potential in therapeutics, diagnostics, and biotechnology.
History
DepositionNov 3, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_CSD / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: BBF-14
C: BBF-14
B: BBF-14_binder4
D: BBF-14_binder4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,45010
Polymers62,5784
Non-polymers8736
Water1267
1
A: BBF-14
B: BBF-14_binder4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,6104
Polymers31,2892
Non-polymers3212
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: BBF-14
D: BBF-14_binder4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,8406
Polymers31,2892
Non-polymers5514
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)41.430, 41.530, 94.000
Angle α, β, γ (deg.)78.426, 82.008, 87.018
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 2 through 13 or resid 15 through 119))
d_2ens_1(chain "C" and (resid 2 through 13 or resid 15 through 119))
d_1ens_2(chain "B" and (resid 4 through 20 or resid 22...
d_2ens_2(chain "D" and (resid 4 through 20 or resid 22...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ens_1GLYGLYTRPTRPAA2 - 132 - 13
d_12ens_1GLYGLYHISHISAA15 - 11915 - 119
d_21ens_1GLYGLYTRPTRPCB2 - 132 - 13
d_22ens_1GLYGLYHISHISCB15 - 11915 - 119
d_11ens_2GLNGLNGLUGLUBC4 - 204 - 20
d_12ens_2LEULEULEULEUBC2222
d_13ens_2LYSLYSLYSLYSBC3434
d_14ens_2LEULEUVALVALBC37 - 5137 - 51
d_15ens_2GLNGLNTHRTHRBC55 - 8655 - 86
d_16ens_2GLUGLUPHEPHEBC89 - 11389 - 113
d_17ens_2ASNASNASNASNBC116116
d_18ens_2PROPROLEULEUBC120 - 129120 - 129
d_19ens_2TRPTRPGLYGLYBC131 - 135131 - 135
d_21ens_2GLNGLNGLUGLUDD4 - 204 - 20
d_22ens_2LEULEULEULEUDD2222
d_23ens_2LYSLYSLYSLYSDD3434
d_24ens_2LEULEUTHRTHRDD37 - 8637 - 86
d_25ens_2GLUGLUPHEPHEDD89 - 11389 - 113
d_26ens_2ASNASNASNASNDD116116
d_27ens_2PROPROLEULEUDD120 - 129120 - 129
d_28ens_2TRPTRPGLYGLYDD131 - 135131 - 135

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.0559970528312, -0.99839498233, -0.00847285852938), (-0.998427679602, 0.0560165213397, -0.00207796580738), (0.00254925069616, 0.00834317651999, -0.999961945639)15.5972166619, 14.3816148213, 75.6330914455
2given(-0.0712794402428, -0.996715599105, -0.038435086821), (-0.997404335168, 0.070829137074, 0.0129547492908), (-0.0101898766678, 0.0392587294958, -0.999177120721)17.8338280902, 13.5281395875, 75.6399791695

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein BBF-14


Mass: 13437.694 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Protein BBF-14_binder4


Mass: 17851.256 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 5 types, 13 molecules

#3: Chemical ChemComp-P6G / HEXAETHYLENE GLYCOL / POLYETHYLENE GLYCOL PEG400


Mass: 282.331 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C12H26O7 / Feature type: SUBJECT OF INVESTIGATION / Comment: precipitant*YM
#4: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#5: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: precipitant*YM
#6: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.51 Å3/Da / Density % sol: 50.93 %
Crystal growTemperature: 289.15 K / Method: vapor diffusion, sitting drop / pH: 6
Details: 0.1 M MES pH 6.0, 0.2 M Na acetate trihydrate, 20% w/v PEG 8000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-3 / Wavelength: 0.9677 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Sep 6, 2024
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9677 Å / Relative weight: 1
ReflectionResolution: 3.1→45.64 Å / Num. obs: 23050 / % possible obs: 90.07 % / Redundancy: 1.9 % / Biso Wilson estimate: 82.94 Å2 / CC1/2: 0.991 / Rmerge(I) obs: 0.129 / Net I/σ(I): 4.15
Reflection shellResolution: 3.1→3.55 Å / Redundancy: 1.6 % / Rmerge(I) obs: 2.559 / Num. unique obs: 1417 / CC1/2: 0.0914 / % possible all: 76.25

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Processing

Software
NameVersionClassification
PHENIX1.21.2_5419refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.1→45.64 Å / SU ML: 0.5273 / Cross valid method: FREE R-VALUE / σ(F): 1.98 / Phase error: 32.3158
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2834 483 4.85 %
Rwork0.2385 9473 -
obs0.2407 9956 90.07 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 109.36 Å2
Refinement stepCycle: LAST / Resolution: 3.1→45.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4036 0 51 7 4094
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00394181
X-RAY DIFFRACTIONf_angle_d0.79775654
X-RAY DIFFRACTIONf_chiral_restr0.0459612
X-RAY DIFFRACTIONf_plane_restr0.007710
X-RAY DIFFRACTIONf_dihedral_angle_d14.93061525
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAX-RAY DIFFRACTIONTorsion NCS0.641442413651
ens_2d_2CBX-RAY DIFFRACTIONTorsion NCS1.04927317229
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.550.33661300.31062692X-RAY DIFFRACTION76.25
3.55-4.470.34281440.25643417X-RAY DIFFRACTION96.82
4.47-45.640.24972090.21013364X-RAY DIFFRACTION97.28
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.0634885893255-0.0979966228995-0.1291123146240.03208686070050.05963985783610.0366721666042-0.5147288178750.569290345254-0.6345923017860.912972145495-1.445692468910.0893942526772-0.6790199703410.1266437873729.07411820647E-60.5397688464330.153769007619-0.1280897970110.495725331771-0.008600731497890.748137431417.09233558540.58164840793752.0148341887
2-0.199299431259-0.0276271060712-0.0577179331720.000730946491070.2210059624830.214477998506-0.3549984190250.6999590541760.374832568776-0.08066690949510.0356773033117-0.501064189237-0.3960262052780.32651524295-2.97938865188E-70.564040292378-0.02591598482610.01600948198770.3675700037270.06526279853150.64931402632223.807847421210.963403326541.6935808883
30.1138902221450.2795819581810.01840197835760.5612111468040.08423814986850.00839389973399-0.162550518093-0.524659370130.081940379503-0.2066160276640.0283402470968-0.1977673812650.09162056549191.49009128626.24648846501E-70.424913881822-0.05663587118580.01534797877560.684296149537-0.07867664262980.59929387155123.117249374813.623934481750.9727267787
40.555620358939-0.2752344541950.7071399038390.5006277567670.3368959133130.289285823363-0.134743938266-0.2502804255920.364852886538-0.05791690883890.07026443207680.141838355446-0.02344102569350.0401896273897-1.93408734114E-70.3783880632590.03234821319970.009744965726940.558370895654-0.1057344383350.59892887587916.355810390613.628167777650.1224506408
50.09458375640470.0103318560393-0.02751427130040.0141684158432-0.161031654868-0.118207229757-0.0006230715009940.1233912118041.044249292220.371644152324-0.404731752658-0.3029270677431.32185973303-0.7298845642835.29923492002E-70.1034321886260.130098729960.1079050953540.372933281893-0.04703603047490.99107671479311.65792024917.650188058447.3624639016
60.5414287591160.6519398939440.6162111726820.4784012129070.257593373294-0.0281452761496-0.2378437580520.4575994622630.440747148190.0060579777602-0.118810418589-0.342643287006-0.248491693195-0.0651236528014-8.62728464987E-80.3627438208060.0714164091081-0.01353186795220.39462976147-0.07139949016960.47893001644414.14647767679.6641655294240.9596787628
7-0.01497415994340.041734413043-0.06580920449070.06514864188820.01924663275110.130285676345-0.551372955940.4718085003290.5365022209960.0748203142752-0.5335272822620.489978538828-0.336595338426-0.0009408638820081.3880889896E-60.5597176446860.0572738713570.1523135329550.641711408021-0.09059757105010.65024620837331.1832542065-0.77589221884639.8763153746
80.195461763228-0.1965676224490.454639555090.5469612621020.6855647660570.03362326876040.35766071784-0.3386831448860.0819886041042-0.1209004519240.02267373308290.000926690452338-0.162494274014-0.05866252564783.67044439879E-70.524045269408-0.0760146202741-0.03602942323970.4318037471340.03148718319150.5476262169034.35531596838-7.7937197033230.0669496885
90.4642114003780.06378328112620.128791331070.9561167818261.110974011260.5968957358060.0511664986806-0.02645455214180.107464519425-0.39647137201-0.1172858261160.438040536128-0.1695877287150.0499450784757-4.01684006577E-70.552542823057-0.0324384275624-0.04950867803880.4149520592140.08740998675250.5012634868310.734752626118-1.4219580036424.6133599844
100.0562122066778-0.255356286107-0.05578749806070.2473818834790.248566651622-0.0801195667547-0.663740549048-0.06289264205820.452553679528-0.9410433923660.395002743260.492785247186-0.504692991577-0.144748065429-4.89395882059E-70.4107217983920.0989479977208-0.2552195667820.5030978557460.009796813033850.7094551386310.3362574044193.6341611143629.5427722161
110.4012635353951.028191454260.07053238629090.7928711475890.09188313650550.566627625563-0.533723302157-0.00791204347344-0.8049609267950.4864931193680.1293696030990.366862870662-0.136004781903-0.0678203451997-1.12134375681E-70.3767329380710.07671687667470.0221268730830.376990445970.001493014891880.5724676527454.48282165182-0.1030457331935.8415828917
120.238692100913-0.07185745586920.1925716894290.0581239767086-0.266481084360.140797450452-0.2715784627581.394277556050.7812818072842.090552631620.137208080688-0.05026938508470.580168479384-0.0721453361872-1.67128353814E-60.7473598524540.0655416619419-0.08512763182130.4781822283980.203769687090.78593932742813.5807138979-16.600902156836.8402216891
130.1668063471350.9323030864520.5337129720380.82142233970.8114458919610.5867759466890.530639649466-1.01320850462-1.286308871610.576539919484-0.3016332521290.2587937970340.5680115209260.266184477965-1.37423870383E-71.50196726392-0.001864264616590.01947270341931.356437681960.4084574779481.3837625902926.4097381781-8.8799512125473.4916542698
140.512961988860.6480132196930.3410859306520.7759457024950.2656230812310.947763515211-0.1643434116810.07663740389050.311417320850.197585845334-0.0621525192172-0.381394291768-0.312440676161-0.4224472329561.16902272143E-60.7507437080160.1364556254880.06959017031920.904533155272-0.06489130378130.63471322001822.75578537395.8189149778863.9523848141
150.0473624263901-0.1862652986960.397284481275-0.535462327760.02180831241240.426132372877-0.4251187226310.2638063488841.03116364405-0.229604537199-0.362812759117-0.4004409741990.1218377981070.1420774496061.82670815919E-60.824419564105-0.160565825380.07630346499940.95127539580.1384517333990.57876740744527.61363783999.3257208127861.2472771528
160.01360383014890.3480523296550.5518084304271.114325543560.2326961776860.8746147565111.40642891622-0.558096425232-0.319003644898-0.0794309705354-0.563064423110.0828838065909-1.56435102516-0.117776810592.92905908016E-70.868901665259-0.1077218972440.1681612726931.60217760330.02790891706760.52427973438627.02420792823.7363794008970.9459098674
17-0.1741291759570.00936188338646-0.324217541093-0.010002607678-0.0418780503387-0.1482429833721.12774256432-1.17681344046-0.870329229938-2.56040586211-3.04411771412-1.454641294511.301450851180.790226935729-8.51775086516E-80.505335496296-0.416697302569-0.4201789178821.63282626045-0.319443477689-0.050756231402420.1459849438-3.5886320261763.3816477135
18-0.1703787783130.6039647401350.281354991134-0.143522784291-0.123114973624-0.0258468653763-0.8514868642080.832396701544-1.14196554559-0.2936100374690.0901519966277-1.05631007233-1.397871153890.6000738741152.26665664313E-62.04551277219-0.04966448772250.07224349154891.81140866466-0.2092705012121.5610895693119.820391321-18.05769141572.04633473036
19-0.243654964110.2519677346610.322772621799-0.0489499231433-0.129134916812-0.08136642137520.2445468866450.6976729299620.650769609597-0.2761661115860.281395057377-1.38517232370.7500619779730.987062781246-1.38658368224E-61.76423311426-0.3193850716090.2507386022912.01234141746-0.3497730626911.2740374935524.1258694919-4.593426952561.29801949649
201.20615681161.17512692105-0.2788085705110.5861942602920.04938077964550.866981408618-0.334122662945-0.163426107529-0.142231807055-0.48393227860.5615943051450.0722287633539-0.318415740076-0.63602725932-1.90225649796E-81.11617987128-0.00264061898923-0.1188278396210.7864952619810.08266169794330.573425570785.96520897625-9.4923047878112.9036764467
210.5153205650010.100998237264-0.2779860207950.1141746654720.6290071486450.501181537140.1912609852760.252239023839-0.0849869720761-0.1404779956040.6217846134711.49727967016-0.485200615012-0.4384180070642.15532604777E-71.92922283022-0.1269630777710.03408464930860.8627357461920.1884950970840.7666248199959.26398750005-12.21754183254.42510392541
22-0.3682173008660.1368779825370.4507237767220.0224891464972-0.0505716569009-0.12819435967-4.26673161819-1.623397005081.47803087295-0.8131284977270.8007235606650.2354461208980.524601982233.436564282271.87986625433E-72.06046460315-0.971303449129-0.1646170312680.329928208441-0.813427195643-0.50728705501816.7520214545-5.6581839505511.2500086242
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 2 through 8 )AA2 - 81 - 7
22chain 'A' and (resid 9 through 23 )AA9 - 238 - 22
33chain 'A' and (resid 24 through 34 )AA24 - 3423 - 33
44chain 'A' and (resid 35 through 66 )AA35 - 6634 - 65
55chain 'A' and (resid 67 through 77 )AA67 - 7766 - 76
66chain 'A' and (resid 78 through 109 )AA78 - 10977 - 108
77chain 'A' and (resid 110 through 122 )AA110 - 122109 - 121
88chain 'C' and (resid 2 through 31 )CC2 - 311 - 30
99chain 'C' and (resid 32 through 66 )CC32 - 6631 - 65
1010chain 'C' and (resid 67 through 85 )CC67 - 8566 - 84
1111chain 'C' and (resid 86 through 109 )CC86 - 10985 - 108
1212chain 'C' and (resid 110 through 122 )CC110 - 122109 - 121
1313chain 'B' and (resid 2 through 50 )BF2 - 501 - 43
1414chain 'B' and (resid 51 through 73 )BF51 - 7344 - 64
1515chain 'B' and (resid 74 through 86 )BF74 - 8665 - 77
1616chain 'B' and (resid 87 through 115 )BF87 - 11578 - 106
1717chain 'B' and (resid 116 through 138 )BF116 - 138107 - 127
1818chain 'D' and (resid 4 through 23 )A0Z0G4 - 231 - 20
1919chain 'D' and (resid 24 through 50 )A0Z0G24 - 5021 - 40
2020chain 'D' and (resid 51 through 86 )A0Z0G51 - 8641 - 73
2121chain 'D' and (resid 87 through 115 )A0Z0G87 - 11574 - 102
2222chain 'D' and (resid 116 through 136 )A0Z0G116 - 136103 - 121

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