Supplemental Data TMC95Based Inhibitor Design Provides Evidence for the Catalytic Versatility of the Proteasome

Supplemental Data

TMC-95-Based Inhibitor Design Provides Evidence

for the Catalytic Versatility of the Proteasome

Michael Groll, Marion Götz, Markus Kaiser, Elisabeth Weyher, and Luis Moroder

Synthesis of the enantiomerically pure 3-fluoro-4-nitrophenylalanine

NO2

F

NO2

F

Br

NBS, AIBN

CCl4 O O

O O

HNO

NO2

F

O O

O O

HNO

NaH, DMF H3NOH

O

NO2

F

conc. HCl

reflux

NH

OH

O

NO2

F

O

Ac2O, NaHCO3,dioxane/H20

Acylase I

pH 7.5, KOHH2NOH

O

NO2

F

L-isomer

Z-OSu, NaHCO3,

dioxane/H2ONH

OH

O

NO2

F

Z

1 2 3

4

567

3-Fluoro-4-nitrobenzylbromide (2): was prepared from commercially available 3-fluoro-

4-nitrotoluene (4.24 g, 27.3 mmol), N-bromosuccinimide (4.86 g, 27.3 mmol) and AIBN

(500 mg) in CCl4 using refluxing conditions for 24 h. The succinimide precipitate was

filtered off and the final product was purified by flash chromatography (petroleum

ether/EtOAc, 5:1) to yield 3.79 g of a yellow oil in 59 % yield; ESI-MS: m/z 235 [M+H]+.

Ethyl 2-(acetylamino)-2-(ethoxycarbonyl)-3-(3’-fluoro-4’-nitrophenyl)propanoate (3)

was prepared according to Vergne et al. 1. Sodium hydride (60 % in paraffin, 0.58 mg, 14.5

mmol) was washed with pentane and suspended in DMF (6 ml). A solution of diethyl

acetamidomalonate (3.45 g, 16.0 mmol) in DMF (15 ml) and a solution of (2) (3.4 g, 14.5

mmol) in DMF (5 ml) were added successively and the reaction mixture was stirred for 4 h

2

at room temperature. The solvent was removed under reduced pressure. The residue was

dissolved in EtOAc and the product was precipitated with increasing amounts of hexane to

give 3.23 g of a white powder in 60% yield; ESI-MS: m/z 371.2 [M+H]+.

(R,S)-3-Fluoro-4-nitrophenylalanine Hydrochloride (4): Compound 3 (3.20 g, 8.60

mmol) was suspended in concentrated HCl (10 ml) and refluxed for 24 h. The volatiles

were evaporated, and the crude residue was co-evaporated three times with toluene and

once with t-butylmethyl ether to furnish 2.10 g of white needles in a 92 % yield; ESI-MS:

m/z 229.0 [M+H]+.

(R,S)-N-Acetyl-3-fluoro-4-nitrophenylalanine (5): Compound 4 (2.10 g, 7.95 mmol) was

dissolved in H2O/dioxane (1:1, 10 ml). The pH of the solution was adjusted to 8-9 by the

addition of solid NaHCO3. Acetic anhydride (2.43 g, 23.9 mmol) was added and the

reaction was stirred at room temperature over night. The volatiles were evaporated and the

residue was redissolved in EtOAc, then washed with KHSO4 (5 %, 3 x 20 ml) and brine (3

x 20 ml) and dried (MgSO4). The solvent was evaporated to give 2.10 g of a white powder

in a 98 % yield; ESI-MS: m/z 271.2 [M+H]+.

(S)-3-Fluoro-4-nitrophenylalanine (6): Compound 5 (315 mg, 1.23 mmol) was

suspended in phosphate buffer (0.1 M, pH 7.5, 10 ml) and then dissolved by the addition of

aqueous KOH (0.1 M, 1 eq). The solution was diluted with additional phosphate buffer

(0.1 M, pH 7.5, 70 ml). Acylase I of Asperigillus melleus (0.55 U/mg, 300 mg) was added

and the reaction was incubated for 20 h at 37 °C. The enzyme was then removed by

Amicon filtration (cut-off >10 kDa). The filtrate was acidified to pH 2-3 using 0.5 N HCl

and the undesired R-isomer was extracted with EtOAc. The pH was increased to 7 using 1

N NaOH. The solvent was removed under vacuum, little H2O was added and the S-isomer

was precipitated over 2 days at 4 °C. Yield: 135 mg (96 %). ESI-MS: m/z 229.2 [M+H]+.

(S)-N-Benzyloxycarbonyl-3-fluoro-4-nitrophenylalanine (7): The amino acid analog 6

(250 mg, 1.10 mmol) was dissolved in H2O/dioxane (1:1, 5 ml). The pH of the solution

was adjusted to 8-9 by the addition of solid NaHCO3. Z-succinimide (273 mg, 2.19 mmol)

was added and the reaction was stirred at room temperature over night. The volatiles were

evaporated and the residue was washed with ether several times to quantitatively give 397

mg of an off white powder; ESI-MS: m/z 362.3 [M+H]+.

3

Synthesis of BIA-1a/b

+ H2N

EDC, HOBt

DMFNH

Boc-Tyr

1. TFA, DCM2. Boc-Asn-OH, EDC, HOBt,DIEA, DMF3. TFA, DCM

NH

Asn-Tyr

NH

Asn-Tyr

NH

OH

O

NO2

F

Z

EDC, HOBt, DIEA, DMF

NH

HN

O

NO2F

ZNH

OHN

OCONH2

OH

NH

HN

O

NO2

ZNH

OHN

OCONH2

OCaCO3, K2CO3

DMF

mol. sieves 4Å45 °C

7 Raney Nickel,H2

MeOH

NH

HN

O

NH2

ZNH

OHN

OCONH2

O

BIA-1b

Boc-Tyr-OH

BIA-1a

Boc-Asn-Tyr-NHCH2CH2CH3: Boc-Tyr-OH (1.00 g, 3.55 mmol) and N-propylamine

(210 mg, 3.55 mmol) were coupled using EDC/HOBt to give 1.13 g of a white powder in a

99 % yield. The crude product was deprotected over 2 h with dilute TFA in CH2Cl2 (20 %,

5 ml) at room temperature. The solvent was evaporated and the crude residue was

triturated with ice-cold t-butyl methyl ether/hexane (2:1). The residue was coupled to Boc-

Asn-OH using the EDC/HOBt coupling conditions. The crude product was redissolved in

EtOAc, however precipitated upon cooling. The precipitate was filtered and washed with

cold EtOAc to give Boc-Asn-Tyr-NHCH2CH2CH3 as a white powder in a 70 % yield. 1H-

NMR (DMSO-d6): 0.78 (t, 3H, propyl-CH3), 1.32 (m, 11H, Boc-tBu + propyl-CH2), 2.28-

2.49 (m, 2H, Asn-CH2), 2.69-2.85 (m, 2H, Tyr-CH2), 2.86-3.01 (m, 2H, N-CH2), 4.16-4.21

(m, 1H, Asn-α-H), 4.24-4.32 (m, 1H, Tyr-α-H), 6.63 (d, 2H, Tyr-Ph), 6.84 (sb, 2H, Asn-

NH2), 6.93 (d, 2H, Tyr-Ph), 7.32 (sb, 1H, NH), 7.71 (sb, 1H, NH), 7.86 (s, 1H, NH), 9.11

(s, 1H, OH).

Z-Phe(3-F,4-NO2)-Asn-Tyr-NHCH2CH2CH3: Boc-Asn-Tyr-NHCH2CH2CH3 was

deprotected using Boc-deprotecting conditions and then coupled to Z-Phe(3-F,4-NO2)-OH

4

(248 mg, 0.551 mmol) using standard EDC/HOBt coupling conditions as described above

to give 279 mg of a white powder in a 76 % yield. ESI-MS: m/z 681.6 [M+H]+.

Macrocyclization to BIA-1a: A solution of Z-Phe(3-F,4-NO2)-Asn-Tyr-NHCH2CH2CH3

(207 mg, 0.304 mmol) in DMF (10 ml) was treated with K2CO3 (210 mg, 1.52 mmol, 5 eq)

and CaCO3 (152 mg, 1.52 mmol, 5 eq) and 4 Å molecular sieves (200 mg) and the mixture

was stirred at 45 °C for 18 h. The solvent was evaporated and the crude product was

purified in several washings with EtOAc to give 89 mg of a white powder in a 44 % yield.

ESI-MS: m/z 661.4 [M+H]+.

Reduction to BIA-1b: BIA-1a (50 mg, 0.076 mmol) was dissolved in MeOH (10 ml) and

one pinch of Raney nickel was added under argon at -5 °C. The mixture was hydrogenated

and the product formation was monitored via HPLC. After 4 h the Raney nickel was

filtered off over celite. The solvent was evaporated and the crude product was purified by

column chromatography (silica gel, MeOH/CH2Cl2, 10 %) to give 15 mg of BIA-1b as a

white powder in a 31 % yield. ESI-MS: m/z 631.3 [M+H]+; TOCSY (DMSO-d6) 0.88 (t,

3H, CH3), 1.49 (m, 2H, CH2CH2CH3), 2.42 (d, 2H, Asn-CH2), 2.12 (d, 1H, Tyr-CH), 2.76

(m, 1H, Tyr-CH), 2.95 (m, 2H, CH2CH2CH3), 3.10 (d, 2H, CH2-Phe(NH2)), 4.19 (t, 1H,

Tyr-α-H), 4.32 (m, 1H, Phe(NH2)-α-H), 4.91-5.01 (m, 2H, Z-CH2), 5.53 (s, 1H, Phe(NH2)-

H-arom), 5.75 (d, 1H, Tyr-NH), 6.11 (d, 1H, Phe(NH2)-H-arom), 6.33 (d, 1H, Phe(NH2)-

H-arom), 6.58 (d, 1H, Tyr-arom), 6.81 (d, 1H, Tyr-arom), 6.90 (s, 2H, Phe-NH2), 7.10 (d,

1H, Tyr-arom), 7.21 (d, 1H, Tyr-arom), 7.22-7.30 (m, 5H, Z-Ph), 7.89 (d, 1H, Asn-NH),

7.92 (t, 1H, NH-propyl), 8.02 (d, 1H, Z-NH).

5

Synthesis of BIA-2a/b

Fmoc-Arg(Pbf)-OHHOBt NH3, EDC

DMFFmoc-Arg(Pbf)-NH2

1. DEA/DMF2. Fmoc-Tyr-OH, EDC, HOBt

DMFFmoc-Tyr-Arg(Pbf)-NH2

Fmoc-Tyr-Arg(Pbf)-NH2

1. DEA/DMF2. Fmoc-Arg(Pbf)-OH, EDC, HOBt3. DEA/DMF

DMFArg(Pbf)-Tyr-Arg(Pbf)-NH2

NH

OH

O

NO2

F

Z

EDC, HOBt, DIEA, DMF

7

NH

HN

O

NO2

ZNH

OHN

ONH2

NH

NH(Pbf)HN

NH

NH(Pbf)HN

O

OHF

CaCO3, K2CO3DMF

mol. sieves 4Å45 °C

NH

HN

O

NO2

ZNH

OHN

ONH2

O

NH

NH(Pbf)HN

NH

NH(Pbf)HN

O

NH

HN

O

NH2

ZNH

OHN

ONH2

O

NH

NHH2N

NH

NHH2N

O 1. Raney Nickel, H22. TFA, CH2Cl2

HOBt⋅⋅⋅⋅NH3: HOBt (4.05 g, 30.0 mmol) was suspended in H2O (30 ml). Ammonia (25 % in

water, 3 ml) was added, and the solid dissolved. The solution was concentrated to 50 % of

the original volume in vacuo. The product was precipitated with acetone, filtered and dried

to give 3.565 g of white needles in a 78 % yield.

Fmoc-Arg(Pbf)-NH2: Fmoc-Arg(Pbf)-OH (2.00 g, 3.08 mmol) and HOBt⋅NH3 (706 mg,

4.62 mmol) were dissolved in DMF (50 ml). EDC (709 mg, 3.70 mmol) was added and the

reaction was stirred at room temperature over night. The solvent was evaporated and the

crude residue was redissolved in EtOAc (100 ml). The organic layer was washed with

aqueous KHSO4 (5 %, 3 x 20 ml), aqueous NaHCO3 (5 %, 3 x 20 ml), brine (3 x 20 ml),

and dried (MgSO4). The solvent was evaporated, and the impurities were removed with

several washings of the solid with diethyl ether to give 1.5 g of a white solid in 75 % yield.

ESI-MS: m/z 648.4 [M+H]+.

Fmoc-Tyr-Arg(Pbf)-NH2: Fmoc-Arg(Pbf)-NH2 (1.1 g, 1.70 mmol) was treated with 20 %

DIEA in DMF (30 ml) for 1 h. The solvent was evaporated and the crude product was

purified by trituration with ice-cold diethyl ether. The deprotected H-Arg(Pbf)-NH2 (600

mg, 1.36 mmol) was coupled to Fmoc-Tyr-OH (547 mg, 1.36 mmol) without further

6

workup using the EDC/HOBt coupling method. Yield: 1.05 g (79 % over two steps). ESI-

MS: m/z 811.4 [M+H]+.

Fmoc-Arg(Pbf)-Tyr-Arg(Pbf)-NH2: Fmoc-Tyr-Arg(Pbf)-NH2 (1.05 g, 1.30 mmol) was

treated with 20 % DIEA in DMF (30 ml) for 1 h. The solvent was evaporated and the crude

product was purified by trituration with ice-cold diethyl ether. The deprotected H-Tyr-

Arg(Pbf)-NH2 (980 mg, 1.67 mmol) was coupled to Fmoc-Arg(Pbf)-OH (1.08 mg, 1.67

mmol) without further workup using the EDC/HOBt coupling method. The product was

purified by column chromatography (silica gel, MeOH/CH2Cl2, 5 % and 2 % Et3N). Yield:

750 mg (47 % over two steps). ESI-MS: m/z 610.4 [(M+2)/2+H]+.

Z-Phe(3-F,4-NO2)-Arg(Pbf)-Tyr-Arg(Pbf)-NH2: Fmoc-Arg(Pbf)-Tyr-Arg(Pbf)-NH2

(750 mg, 0.615 mmol) was treated with 20 % DIEA in DMF (20 ml) for 1 h. The solvent

was evaporated and the crude product was purified by trituration with ice-cold diethyl

ether. The deprotected H-Arg(Pbf)-Tyr-Arg(Pbf)-NH2 (610 mg, 0.612 mmol) was coupled

to Z-Phe(3-F,4-NO2)-OH (222 mg, 0.612 mmol) without further workup using the

EDC/HOBt coupling method. The product was purified by column chromatography (silica

gel, MeOH/CH2Cl2, 7 % and 2 % Et3N). Yield: 704 mg (85 % over two steps). ESI-MS:

m/z 671.6 [(M+2)/2+H]+.

Macrocyclization to BIA-2a: A solution of Z-Phe(3-F,4-NO2)-Arg(Pbf)-Tyr-Arg(Pbf)-

NH2 (704 mg, 0.525 mmol) in DMF (10 ml) was treated with K2CO3 (363 mg, 2.62 mmol,

5 eq) and CaCO3 (263 mg, 2.62 mmol, 5 eq) and 4 Å molecular sieves (300 mg) and the

mixture was stirred at 45 °C for 4 days, however some starting material remained. The

reaction mixture was cooled down and filtered through a pad of celite (EtOAc wash) and

concentrated in vacuo. The crude red residue was purified by column chromatography

(silica gel, MeOH/CH2Cl2, 10 % and 1 % Et3N, Rf = 0.2). Yield: 180 mg (26 %). ESI-MS:

m/z 1321.8 [M+H]+.

Reduction to BIA-2b: The macrocycle BIA-2a (150 mg, 0.114 mmol) was dissolved in

MeOH (10 ml) and one scoop of Raney nickel was added under argon at -5 °C. The

mixture was hydrogenated and the product formation was monitored via HPLC. After 24 h

the Raney nickel was filtered off over celite. The solvent was evaporated and the crude

product was purified by column chromatography (silica gel, MeOH/CH2Cl2 10 %) to give

60 mg of BIA-2b as a white powder in a 41 % yield of crude material. The crude

macrocycle was deprotected with 50 % TFA in CH2Cl2 (5 ml) for 1 h at room temperature.

7

The solvent was evaporated and the product precipitated with ice-cold tert-butyl methyl

ether/hexane (2:1) and then purified by semi preparative HPLC to give 3 mg of a white

powder in a 6 % yield. ESI-MS: m/z 394.4 [(M+2)/2+H]+.

Synthesis of BIA-3a

The linear precursor Z-Phe(3-F,4-NO2)-Arg(Pbf)-Tyr-Arg(Pbf)-Ala-Gly-NH2 was

synthesized on a 0.31 mmol scale of Sieber Amide Resin (loading 0.62 mmol/g) from

NovaBiochem and Fmoc/HOBT/HATU/diisopropylethylamine chemistry with 20 %

diethylamine in DMF deprotection. The Pbf-protected arginine residues were coupled

twice after finding incomplete coupling by the TNBS test. All reagents were coupled with

4 eq except for the final Z-Phe(4-NO2,3-F)-OH, which was coupled with 1.5 eq due to

material shortage. Cleavage off the resin was accomplished with 1 % TFA in CH2Cl2 for 5

min with subsequent precipitation into ice-cold tert-butyl methyl ether/hexane (2:1). This

procedure was repeated 5 times to give 0.38 mg of the peptide as a white solid. ESI-MS:

m/z 1469.6 [M+H]+. A solution of this crude material (377 mg, 0.257 mmol) in DMF (20

ml) was treated with K2CO3 (178 mg, 1.29 mmol, 5 eq) and CaCO3 (129 mg, 1.29 mmol, 5

eq) and 4 Å molecular sieves (200 mg) and the mixture was stirred at 45 °C for 4 days,

however 30 % of the starting material remained. The reaction mixture was cooled down

and filtered through a pad of celite (EtOAc wash) and concentrated in vacuo. The crude red

residue was purified by column chromatography (silica gel, MeOH/CH2Cl2 10 % and 1 %

Et3N, Rf = 0.45). Yield: 30 mg (8 %). ESI-MS: m/z 1321.8 [M+H]+

The macrocycic peptide was deprotected with 50 % TFA in CH2Cl2 (5 ml) for 1 h at room

temperature. The solvent was evaporated and the product precipitated with ice-cold tert-

butyl methyl ether/hexane (2:1) to give 8 mg of a white powder in a 41 % yield. ESI-MS:

m/z 1059.8 [M+TFA+H]+.

Kinetic Assays

Kinetic assays were performed on a single cell fluorometer using with a total DMSO

concentration below 1 %. The chymotryptic activity was measured in 20 mM HEPES, 0.5

mM EDTA, 0.037 % SDS, pH 7.8 at 37 °C, with 4.92 µM Suc-Leu-Leu-Val-Tyr-AMC as

fluorogenic substrateand 0.5 nM enzyme concentration at 380 nm excitation and 460 nm

emission. The tryptic activity was measured in 50 mM Tris, 1 mM EDTA, 100 mM NaCl,

8

pH 7.5 at 37 °C, with 19.42 µM Bz-Phe-Val-Arg-AMC as the fluorogenic substrate, and

2.0 nM enzyme concentration at 380 nm excitation and 460 nm emission. The PGPH

activity was measured in 50 mM Tris, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, pH 7.5 at

37 °C, with 39 µM Z-Leu-Leu-Glu-βNa as the fluorogenic substrate, and 2.0 nM enzyme

concentration at 345 nm excitation and 425 nm emission. Inhibitors were dissolved in

DMSO and used at 1-300 mM concentration in the assays. The Ki values were derived as

described previously 2, 3.

X-ray crystallography

Co-crystallisation

Crystals of the 20S proteasome from S. cerevisiae were grown in hanging drops at 24°C as

has been described4 and incubated for 60 min with compound BIA20 and BIA32,

respectively. The protein concentration used for crystallization was 40mg/ml in Tris-HCl

(10 mM, pH 7.5) and EDTA (1 mM). The drops contained 3 µl of protein and 2 µl of the

reservoir solution, containing 30 mM of magnesium acetate, 100 mM of morpholino-

ethane-sulphonic acid (pH 7.2) and 10% of MPD.

The space group belongs to P21 with cell dimensions of a=136 Å, b=300 Å, c=144 Å and

β �=113°. Data to 3.1 Å for the CP:BIA20 and to 2.8 Å for the CP:BIA32-complex were

collected using synchrotron radiation with λ �= 1.05 Å on the BW6-beamline at DESY/

Hamburg/Germany. Crystals were soaked in a cryoprotecting buffer (30% MPD, 20 mM of

magnesium acetate, 100 mM of morpholino-ethane-sulfonic acid pH 6.9) and frozen in a

stream of liquid nitrogen gas at 90 K (Oxford Cryo Systems). X-ray intensities were

evaluated by using the MOSFILM program package (version 6.1) and data reduction was

performed with CCP4 5. The anisotropy of diffraction was corrected by an overall

anisotropic temperature factor by comparing observed and calculated structure amplitudes

using the program CNS 6. Electron density was improved by averaging and back

transforming the reflections 10 times over the twofold noncrystallographic symmetry axis

using the program package MAIN 7. Conventional crystallographic rigid body, positional

and temperature factor refinements were carried out with CNS 6 using the yeast 20S

proteasome structure as starting model 8. For model building the program MAIN was used.

Modelling experiments were performed using the coordinates of yeast 20S proteasome

with the program MAIN 7.

9

Table. Data collection and refinement statistics

CP:BIA-1a CP:BIA-2a Crystal parameters Space group P21 P21 Cell constants (one molecule / AUa)

a=135.5Å; b=301 Å, c=144.3 Å β=113.0°

a=136.4Å; b=301 Å, c=144.8 Å β=113.2°

Data collection Beamline BW6, DESY BW6, DESY Wavelength (Å) 1.05 1.05 Resolution range (Å)b 99-2.81 (2.85-2.81) 99-3.1 (3.12-3.07) No. observations 3680171 2920847 No. unique reflectionsc 252489 192705 Completeness (%)b 97.6 (85.8) 97.4 (98.7) Rmerge (%)b, d 6.2 (33.1) 8.1 (39.4) I/σ (I)b 26.3 (1.9) 18.2 (1.2) Refinement (CNS) Resolution range (Å) 15-2.81 15-3.1 No. reflections working set 238485 176430 No. reflections test set 12489 9291 No. non hydrogen 49644 2598 Solvent water 1070 Inhibitor (non hydrogen) 96 116 Rwork/Rfree (%)f 21.7 / 24.1 20.1 / 23.6 rmsd bond lengths (Å) / (°)g 0.007 / 1.3 0.007 / 1.3

a Asymmetric unit. b The values in parentheses of resolution range, completeness, Rmerge and I/σ (I) correspond to the last resolution

shell. c Friedel pairs were treated as different reflections. d Rmerge(I) = ΣhklΣj | I(hkl)j - I(hkl)] |/[Σhkl Ihkl , where I(hkl)j is the jth measurement of the intensity of reflection

hkl and <I(hkl)> is the average intensity. e Figure of merit = <ΣαP(α)eix/ΣαP(α), after density modification, where α is the phase and P(α) is the phase

probability distribution. f R = Σhkl | |Fobs| - |Fcalc| |/Σhkl |Fobs|, where Rfree

9 is calculated without a sigma cutoff for a randomly chosen 5% of reflections, which were not used for structure. refinement, and Rwork is calculated for the remaining reflections.

g Deviations from ideal bond lengths/angles 10. Supplemental References

1. Vergne, C., Bois-Choussy, M., Ouazzani, J., Beugelmans, R. & Zhu, J.

Chemoenzymatic Synthesis of Enantiomerically pure 4-Fluoro-3-Nitro and 3-Fluoro-4-nitro phenylalanine. Tetrahedron: Asymmetry 8, 391-398 (1997).

2. Kaiser, M. et al. Binding mode of TMC-95A analogues to eukaryotic 20S proteasome. ChemBioChem 5, 1256-66 (2004).

3. Kaiser, M. et al. TMC-95A analogues with endocyclic biphenyl ether group as proteasome inhibitors. Chem. Biodiv. 1, 161-173 (2004).

10

4. Groll, M. & Huber, R. Purification, crystallization and X-ray analysis of the yeast 20S proteasomes. Methods Enzymol. 398, 329-336 (2005).

5. Lesslie, A. G. Mosfilm user guide, mosfilm version 5.2. MRC Laboratory of Molecular Biology, Cambrige, UK. (1994).

6. Brünger, A. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1, 905-921 (1998).

7. Turk, D. Improvement of a programm for molecular graphics and manipulation of electron densities and its application for protein structure determination. Thesis, Technische Universitaet Muenchen (1992).

8. Groll, M. et al. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386, 463-71 (1997).

9. Brünger, A. T. Assessment of phase accuracy by cross validation: the free R value. Methods and applications. Acta Crystallogr D Biol Crystallogr 49, 24-36 (1993).

10. Engh, R. & Huber, R. Accurate bond and angles parameters for X-ray protein structure refinement. Acta Cryst. A47, 392-400 (1991).