in vivo protein crystallization opens new routes in ... · nature methods in vivo protein...

Nature Methods

In vivo protein crystallization opens new routes in structural biology Rudolf Koopmann, Karolina Cupelli, Lars Redecke, Karol Nass, Daniel DePonte, Thomas A White,

Francesco Stellato, Dirk Rehders, Mengning Liang, Jakob Andreasson, Andrew Aquila, Sasa Bajt, Miriam

Barthelmess, Anton Barty, Michael J Bogan, Christoph Bostedt, Sébastien Boutet, John D Bozek, Carl

Caleman, Nicola Coppola, Jan Davidsson, R Bruce Doak, Tomas Ekeberg, Sascha W Epp, Benjamin Erk,

Holger Fleckenstein, Lutz Foucar, Heinz Graafsma, Lars Gumprecht, Janos Hajdu, Christina Y Hampton,

Andreas Hartmann, Robert Hartmann, Günter Hauser, Helmut Hirsemann, Peter Holl, Mark S Hunter,

Stephan Kassemeyer, Nils Kimmel, Richard A Kirian, Lukas Lomb, Filipe R N C Maia, Andrew V Martin,

Marc Messerschmidt, Christian Reich, Daniel Rolles, Benedikt Rudek, Artem Rudenko, Ilme Schlichting,

Joachim Schulz, M Marvin Seibert, Robert L Shoeman, Raymond G Sierra, Heike Soltau, Stephan Stern,

Lothar Strüder, Nicusor Timneanu, Joachim Ullrich, Xiaoyu Wang, Georg Weidenspointner, Uwe

Weierstall, Garth J Williams, Cornelia Wunderer, Petra Fromme, John C H Spence, Thilo Stehle, Henry N

Chapman, Christian Betzel & Michael Duszenko

Supplementary Figure 1 Characterization of TbCatB isolated from in vivo crystals. Supplementary Figure 2 Experimental setup of serial femtosecond crystallography at LCLS. Supplementary Figure 3 Quality measures of TbCatB diffraction dataset obtained at LCLS. Supplementary Table 1 Diffraction data statistics by resolution shell.

Supplementary Table 2 Data collection and refinement statistics for re-crystallized TbCatB analyzed at SLS.

Supplementary Table 3 Electron and photon beam parameters for SFX experiment at LCLS. Supplementary Note

Nature Methods: doi:10.1038/nmeth.1859

Supplementary Figure 1

Characterization of TbCatB isolated from in vivo crystals.

Supplementary Figure 1: Characterization of TbCatB isolated from in vivo crystals. (a)

Western blot analysis: 1, commercial Cathepsin B from Bos Taurus; 2, TbCatB solubilised

from isolated in vivo crystals; (b) Specific activity of TbCatB solubilised from isolated in vivo

crystals. Different amounts of TbCatB were tested using a commercial CatB activity assay

(online methods). As a control, enzyme activity was analyzed in the presence of 50 M CatB

specific inhibitor as well as after boiling the enzyme solution for 5 min. (c) Deglycosylation of

solubilised TbCatB using different amounts of PNGase F (15 % SDS gel, silver staining).



Experimental setup of serial femtosecond crystallography at LCLS

Supplementary Figure 2: Experimental setup of serial femtosecond crystallography at LCLS.

Each single 70 fs X-ray pulse focussed to 2.5 3 m2 at the FWHM hits the 4 m diameter

water jet containing the in vivo crystals injected perpendicular to the FEL beam. The

diffraction pattern of each single crystal was recorded by a high frame rate pnCCD detector1

operating at the 60 Hz repetition rate of the X-ray pulses.



Quality measures of TbCatB diffraction dataset obtained at LCLS

Supplementary Figure 3: Quality measures of TbCatB diffraction dataset obtained at LCLS.

(a) NZ-plots, (b) L-test, and (c) Wilson plot for the SFX data of TbCatB in vivo crystals

recorded at LCLS. NZ and L-plots were drawn using the program xtriage of the Phenix

package2, the Wilson plot was calculated by the program truncate of the ccp4i suite3.


Supplementary Table 1: Quality indicators for the resolution shells of the partial dataset

collected from TbCatB in vivo crystals using the SFX method at LCLS.

Resolution shell (Å)

No. of unique reflections

Multiplicity Completeness

(%) Merged

I/

43.468-15.748 63 11.9 88.3 7.06

15.748-12.600 66 18.2 95.6 2.35

12.600-11.037 67 27.0 97.1 2.44

11.037-10.042 56 19.0 87.1 2.45

10.042-9.329 59 18.1 91.2 2.10

9.329-8.784 55 15.1 90.4 1.98

8.784-8.348 49 10.8 82.3 1.88

8.348-7.986 52 11.3 83.8 1.81

7.986-7.681 37 4.2 80.4 0.75

7.681-7.417 16 1.6 37.0 1.36

Total 514 13.72 83.3 2.63


Supplementary Table 2: X-ray data collection and refinement statistics for re-crystallized

TbCatB analyzed at the Swiss Light Source (SLS).

Data collection

Space group P21

Cell dimensions

a, b, c (Å) 53.91, 75.49, 75.60

α, β, γ () 90.0, 104.8, 90.0

Resolution (Å) 48.7 - 2.55 (2.62 - 2.55)

Rmeas 15.3 (47.2)

I/I 8.60 (2.69)

Completeness (%) 97.2 (98.4)

Redundancy 2.2 (2.1)

Refinement

Resolution (Å) 48.0 - 2.55

No. reflections 17,772

Rwork / Rfree 20.2 / 24.3

No. atoms

Protein 4,020

Carbohydrate 56

Water 54

B-factors (Å2)

Protein 10.1

Carbohydrate 25.3

Water 21.5

R.m.s. deviations

Bond lengths (Å) 0.006

Bond angles () 0.600

Values in parentheses refer to the highest resolution shell.


Supplementary Table 3: Electron and photon beam parameters for SFX experiment of

TbCatB in vivo crystals at LCLS.

Parameter Mean Standard deviation

Electron energy [MeV]a 6,710.8 8.4

Number of electronsa 1.56 e09 2.99 e07

Charge [nC]a 0.249 0.005

Peak current after second bunch compressor [A]a 3,613.05 434.59

X-ray pulse duration [fs]a 67.4 -

Electron pulse duration [fs]a 70.1 9.1

Photon energy [mJ]a 2.13 0.18

Number of photonsa 6.7 e12 5.5 e11

Photon energy [eV]a 1,995.4 5.4

Photon wavelength [Å]a 6.21 0.02

Wavelength jitter [%] 0.27 -

Peak X-ray power [GW]a 30.5 2.5

Beam intensity in focus [W cm-2]b 5.1 e18 - a) Varies from shot to shot; b) Focus 2 x 3 m2


Supplementary Note

Analysis of protein content from solubilised in vivo crystals. Isolated and purified in vivo

crystals were solubilised in sodium acetate buffer at pH 3.5. As shown by Western blot

analysis, a polyclonal Cathepsin B specific antibody detected the solubilised protein

(Supplementary Fig. 1a), suggesting that the expressed TbCatB protein is a major constituent

of the in vivo crystals. This was confirmed by a commercial CatB activity assay containing a

flourogenic dipeptide substrate and a specific CatB inhibitor (Supplementary Fig. 1b) as well

as by mass spectrometry performed on the trypsin digested gel bands (data not shown). N-

terminal sequencing identified Glu63 of the propeptide as the first protein residue within the in

vivo crystals, confirming the persistence of a part of the propeptide that is normally auto-

catalytically cleaved in the lysosome4. Two protein bands corresponding to molecular weights

of approx. 30 and 34 kDa were detected by SDS-PAGE analysis (Supplementary Fig. 1c),

which converged after de-glycosylation treatment to a single protein band at about 30 kDa.

Since the identified amino acid sequence of TbCatB contains two N-glycosylation consensus

motifs, one within the remnant pro-region at Asn76 and another at Asn216 of the mature

protein sequence, different glycosylation states of TbCatB in the in vivo crystals are indicated.

Crystal contacts within in vitro crystallized TbCatB. The essential crystal contacts

maintaining the crystal lattice primarily involve the hydrophobic patches of two equivalent

anti-parallel -helices within the two neighbouring monomers A and B, comprising residues

Arg84 to Pro95 of the propeptide sequence of each molecule. The patch is flanked by a region

of hydrogen bonds, polar contacts, and hydrophobic interactions that are mediated by the

monomers A, B and C (Fig. 3b). In total, these interactions bury a surface area of approx.

1,730 Å2, thus comprising the largest continuous buried surface area in the crystal packing.

Therefore, the presence of the C-terminal residues of the propeptide of TbCatB might

influence also the crystal formation in vivo.

Differences of TbCatB to human Cathepsin B. Although the structure of TbCatB exhibits

the characteristic papain/CatB fold, there are distinct differences to human CatB (PDB ID:

1GMY)5 that are particularly important to consider for rational drug discovery investigations.

Both TbCatB structures still contain an additional segment of the pro-peptide that is in

principle predicted to be completely cleaved during protein maturation under acidic

conditions4. This remnant pro-peptide end, located near the S2-pocket and reducing the


accessibility of the catalytic cleft, may influence and trigger the binding of ligands.

Furthermore, some residues of the S2-pocket itself, which is considered to be important for

substrate specificity of the protease6, differ to the corresponding residues of the human protein.

For example, HsCatB contains glutamate at position 324 and tyrosine at position 154 close to

the bottom of the pocket, while TbCatB has a glycine and an aspartic acid residue at the

corresponding position 328 and 166, respectively. This modification results in an enlarged

pocket for TbCatB, allowing substrates with larger and non-polar amino acid side chains to

bind. These significant structural differences can be used to guide the design of a specific

inhibitor, since the remaining active site is highly conserved.

1. Strüder, L. et al. Nuc. Inst. Meth. Phys. Res. A 614, 483-496 (2010).

2. Adams, P.D. et al. Acta Crystallogr. D Biol. Crystallogr. 58, 1948-1954 (2002).

3. Collaborative Computational Project, Number 4, Acta Crystallogr. D Biol. Crystallogr.

50, 760-763 (1994).

4. Mackey, Z.B., O'Brien, T.C., Greenbaum, D.C., Blank, R.B. & McKerrow, J.H. J. Biol.

Chem. 279, 48426-48433 (2004).

5. Greenspan, P.D. et al. J. Med. Chem. 44, 4524-4534 (2001).

6. Sajid, M. & McKerrow, J.H. Mol. Biochem. Parasitol. 120, 1-21 (2002).


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