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Basic information

Entry
Database: PDB / ID: 8vge
TitleCrystal structure of an engineered conformationally rigid anti-Tryptase Fab variant E104.v1.4DS.A114F
Components
  • Fab E104.v1.4DS.A114F heavy chain
  • Fab E104.v1.4DS.A114F light chain
KeywordsIMMUNE SYSTEM / antibody fragment / fab / protein engineering / tryptase
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.01 Å
AuthorsKung, J.E. / Sudhamsu, J.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: bioRxiv / Year: 2024
Title: Disulfi de constrained Fabs overcome target size limitation for high-resolution single-particle cryo-EM.
Authors: Jennifer E Kung / Matthew C Johnson / Christine C Jao / Christopher P Arthur / Dimitry Tegunov / Alexis Rohou / Jawahar Sudhamsu /
Abstract: High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure ...High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure determination of large proteins and their complexes, but a vast majority of proteins that underlie human diseases are small (< 50 kDa) and usually beyond its reach due to low signal-to-noise images and difficulties in particle alignment. Current strategies to overcome this problem increase the overall size of small protein targets using scaffold proteins that bind to the target, but are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the scaffolds. Here we present an iteratively engineered molecular design for transforming Fabs (antibody fragments), into conformationally rigid scaffolds (Rigid-Fabs) that, when bound to small proteins (~20 kDa), can enable high-resolution structure determination using cryo-EM. This design introduces multiple disulfide bonds at strategic locations, generates a well-folded Fab constrained into a rigid conformation and can be applied to Fabs from various species, isotypes and chimeric Fabs. We present examples of the Rigid Fab design enabling high-resolution (2.3-2.5 Å) structures of small proteins, Ang2 (26 kDa) and KRAS (21 kDa) by cryo-EM. The strategies for designing disulfide constrained Rigid Fabs in our work thus establish a general approach to overcome the target size limitation of single particle cryo-EM.
History
DepositionDec 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: Fab E104.v1.4DS.A114F light chain
H: Fab E104.v1.4DS.A114F heavy chain


Theoretical massNumber of molelcules
Total (without water)48,9272
Polymers48,9272
Non-polymers00
Water3,747208
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3520 Å2
ΔGint-21 kcal/mol
Surface area18810 Å2
MethodPISA
Unit cell
Length a, b, c (Å)115.099, 115.099, 137.830
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122
Space group name HallP612(x,y,z+5/12)
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/6
#3: y,-x+y,z+5/6
#4: -y,x-y,z+1/3
#5: -x+y,-x,z+2/3
#6: x-y,-y,-z
#7: -x,-x+y,-z+2/3
#8: -x,-y,z+1/2
#9: y,x,-z+1/3
#10: -y,-x,-z+5/6
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+1/6

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Components

#1: Antibody Fab E104.v1.4DS.A114F light chain


Mass: 23603.307 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody Fab E104.v1.4DS.A114F heavy chain


Mass: 25323.541 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 208 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.69 Å3/Da / Density % sol: 54.33 %
Crystal growTemperature: 292 K / Method: vapor diffusion / Details: 0.1 M sodium citrate, pH 4.5, 20% PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 14, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 2.01→46.87 Å / Num. obs: 36470 / % possible obs: 98.58 % / Redundancy: 18.8 % / Biso Wilson estimate: 54.7 Å2 / CC1/2: 1 / Rsym value: 0.08241 / Net I/σ(I): 26.57
Reflection shellResolution: 2.01→2.082 Å / Num. unique obs: 69671 / CC1/2: 0.808 / Rsym value: 1.578

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Processing

Software
NameVersionClassification
PDB_EXTRACTdata extraction
PHENIX1.20rc3-4406_finalrefinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.01→46.87 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 26.741
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.234 2000 5.56 %
Rwork0.209 33959 -
obs0.2104 35959 98.6 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 53.8 Å2
Refinement stepCycle: LAST / Resolution: 2.01→46.87 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3203 0 0 208 3411
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00233360
X-RAY DIFFRACTIONf_angle_d0.58854589
X-RAY DIFFRACTIONf_chiral_restr0.0445533
X-RAY DIFFRACTIONf_plane_restr0.0039583
X-RAY DIFFRACTIONf_dihedral_angle_d12.20671215
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.01-2.060.35661420.32172417X-RAY DIFFRACTION99.61
2.06-2.120.35891410.33452403X-RAY DIFFRACTION99.8
2.12-2.180.31051420.28672403X-RAY DIFFRACTION100
2.18-2.250.40991300.37892181X-RAY DIFFRACTION90.56
2.25-2.330.3591290.30452215X-RAY DIFFRACTION91.1
2.33-2.420.27871440.23422424X-RAY DIFFRACTION99.96
2.42-2.530.23261420.22252433X-RAY DIFFRACTION100
2.53-2.670.23541440.22882431X-RAY DIFFRACTION99.84
2.67-2.830.28051440.23322439X-RAY DIFFRACTION100
2.83-3.050.24051450.22512458X-RAY DIFFRACTION100
3.05-3.360.23741440.20622464X-RAY DIFFRACTION99.96
3.36-3.840.22591470.19242486X-RAY DIFFRACTION99.43
3.84-4.840.1541480.15332526X-RAY DIFFRACTION99.85
4.84-46.870.21691580.17982679X-RAY DIFFRACTION99.93
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)
15.12551.148-1.23322.8699-4.13647.8626-0.0822-0.1066-0.34020.12420.14220.10960.1788-0.1865-0.00480.30930.02460.02410.22-0.08930.42750.424-60.90311.21
28.5094-1.6446-1.9391.85930.19925.7223-0.23830.1346-0.86270.04680.37741.00240.4315-1.2195-0.11620.2862-0.01270.08850.44320.00460.6539-12.454-54.22914.748
31.4194-0.5879-0.19237.23950.02264.0218-0.00380.0439-0.3375-0.22380.04340.51320.1629-0.3189-0.05960.26660.0292-0.01290.3464-0.07470.4942-7.816-55.0775.107
48.753-2.1982-4.75825.38492.55343.45460.16460.3122-0.0377-0.45670.07690.3464-0.2775-0.2996-0.32390.26440.0268-0.0210.2681-0.06370.35580.237-54.4835.068
53.77312.2735-1.89982.38280.30953.1157-0.1223-0.6604-0.89451.2873-0.00551.1496-0.0515-0.56570.3590.6020.07040.29490.59130.03010.7384-12.51-49.84420.623
62.57521.7185-0.56331.3009-0.04460.96-0.35260.914-0.0123-0.48510.6114-0.7323-0.2310.0752-0.32280.5183-0.09940.22210.5223-0.09920.618921.639-41.4520.022
75.65435.8641.1867.23531.86361.40140.00090.2491-0.8780.01170.4641-2.0761-0.11980.1659-0.37770.4157-0.04660.17630.5495-0.20760.673926.431-44.2332.679
88.60351.9601-1.25143.2354-0.3413.30340.18810.1481-0.40450.27310.3158-0.56750.5477-0.1027-0.14220.36280.06260.06870.3519-0.11390.439321.963-44.9163.749
94.08610.0286-0.9840.0428-0.22471.3572-0.0750.5276-0.2283-0.50640.6231-1.7163-0.23230.4883-0.07540.4336-0.12990.29190.674-0.27231.008233.785-39.0681.087
106.798-0.82470.97444.1122-1.25685.3736-0.05340.35380.2097-0.4361-0.0810.297-0.0969-0.18010.13350.23940.0706-0.01040.2751-0.05520.2519-7.582-31.56311.899
115.1004-0.0327-1.36361.40450.07630.9716-0.04480.0159-0.3354-0.1447-0.0094-0.0087-0.1597-0.02780.07040.29750.0558-0.01540.2903-0.03770.2359-2.562-34.61614.184
122.2904-0.7208-1.98673.45112.09616.58890.01291.00030.2388-0.818-0.16080.3081-0.7216-0.59240.13340.5744-0.0068-0.00880.73910.0070.338813.915-30.644-0.013
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN L AND RESID 3:24 )L3 - 24
2X-RAY DIFFRACTION2( CHAIN L AND RESID 25:38 )L25 - 38
3X-RAY DIFFRACTION3( CHAIN L AND RESID 39:75 )L39 - 75
4X-RAY DIFFRACTION4( CHAIN L AND RESID 76:91 )L76 - 91
5X-RAY DIFFRACTION5( CHAIN L AND RESID 92:103 )L92 - 103
6X-RAY DIFFRACTION6( CHAIN L AND RESID 104:130 )L104 - 130
7X-RAY DIFFRACTION7( CHAIN L AND RESID 131:152 )L131 - 152
8X-RAY DIFFRACTION8( CHAIN L AND RESID 153:176 )L153 - 176
9X-RAY DIFFRACTION9( CHAIN L AND RESID 177:212 )L177 - 212
10X-RAY DIFFRACTION10( CHAIN H AND RESID 3:40 )H3 - 40
11X-RAY DIFFRACTION11( CHAIN H AND RESID 41:150 )H41 - 150
12X-RAY DIFFRACTION12( CHAIN H AND RESID 151:218 )H151 - 218

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