8A67

Branched Lys48- and Lys63-linked tri-ubiquitin (K48-K63-Ub3) in complex with matured synthetic nanobody NbSL3.3Q (3rd generation)

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE ID23-2
Synchrotron site	ESRF
Beamline	ID23-2
Temperature [K]	100
Detector technology	PIXEL
Collection date	2022-06-09
Detector	DECTRIS PILATUS3 2M
Wavelength(s)	0.87313
Spacegroup name	P 1
Unit cell lengths	57.081, 58.243, 61.662
Unit cell angles	78.88, 67.94, 80.16

Refinement procedure

Resolution	52.590 - 1.860
R-factor	0.1893
Rwork	0.186
R-free	0.24520
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	7nbb
RMSD bond length	0.002
RMSD bond angle	0.503
Data reduction software	autoPROC
Data scaling software	XSCALE
Phasing software	PHENIX
Refinement software	PHENIX (1.20.1_4487)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	56.580	2.090
High resolution limit [Å]	1.860	1.860
Number of reflections	36500	1826
<I/σ(I)>	4.1
Completeness [%]	88.7
Redundancy	3.2
CC(1/2)	0.987	0.735

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.5	293.15	Protein concentrated to 14.5 mg/ml in 20 mM HEPES pH 7.5, 150 mM NaCl. Mixed 200 nl protein with 100 nl mother liquor (0.1 M HEPES pH 7.5, 10% 2-propanol, 20% PEG4000). Crystals harvested and cryo-protected with Mother liquor supplemented with 30% glycerol.