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+anno_start	anno_end	anno_text	entity_type	sentence	section
+40	64	autocatalytic activation	ptm	A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome	TITLE
+72	86	20S proteasome	complex_assembly	A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome	TITLE
+18	32	20S proteasome	complex_assembly	Biogenesis of the 20S proteasome is tightly regulated.	ABSTRACT
+15	26	propeptides	structure_element	The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly.	ABSTRACT
+42	53	active-site	site	The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly.	ABSTRACT
+54	64	threonines	residue_name	The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly.	ABSTRACT
+69	86	autocatalytically	ptm	The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly.	ABSTRACT
+68	87	strict conservation	protein_state	However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic.	ABSTRACT
+91	100	threonine	residue_name	However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic.	ABSTRACT
+12	23	mutagenesis	experimental_method	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+25	46	X-ray crystallography	experimental_method	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+51	69	biochemical assays	experimental_method	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+86	91	Lys33	residue_name_number	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+129	139	propeptide	structure_element	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+161	165	Thr1	residue_name_number	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+218	223	Asp17	residue_name_number	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+238	253	catalytic triad	site	Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad.	ABSTRACT
+0	12	Substitution	experimental_method	Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.	ABSTRACT
+16	20	Thr1	residue_name_number	Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.	ABSTRACT
+24	27	Cys	residue_name	Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.	ABSTRACT
+58	63	Lys33	residue_name_number	Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.	ABSTRACT
+68	79	inactivates	protein_state	Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.	ABSTRACT
+84	94	proteasome	complex_assembly	Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome.	ABSTRACT
+11	18	Thr1Ser	mutant	Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates.	ABSTRACT
+19	25	mutant	protein_state	Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates.	ABSTRACT
+29	35	active	protein_state	Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates.	ABSTRACT
+72	81	wild type	protein_state	Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates.	ABSTRACT
+125	129	Ser1	residue_name_number	Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates.	ABSTRACT
+72	92	propeptide autolysis	ptm	This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.	ABSTRACT
+147	157	proteasome	complex_assembly	This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.	ABSTRACT
+170	188	threonine protease	protein_type	This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease.	ABSTRACT
+5	15	proteasome	complex_assembly	 The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear.	ABSTRACT
+54	72	threonine protease	protein_type	 The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear.	ABSTRACT
+85	95	proteasome	complex_assembly	Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity.	ABSTRACT
+96	107	active site	site	Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity.	ABSTRACT
+4	32	20S proteasome core particle	complex_assembly	The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells.	INTRO
+34	36	CP	complex_assembly	The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells.	INTRO
+49	71	non-lysosomal protease	protein_type	The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells.	INTRO
+75	85	eukaryotic	taxonomy_domain	The 20S proteasome core particle (CP) is the key non-lysosomal protease of eukaryotic cells.	INTRO
+20	21	α	protein	Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder.	INTRO
+42	52	β subunits	protein	Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder.	INTRO
+72	82	heptameric	oligomeric_state	Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder.	INTRO
+83	88	rings	structure_element	Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder.	INTRO
+130	145	hollow cylinder	structure_element	Its seven different α and seven different β subunits assemble into four heptameric rings that are stacked on each other to form a hollow cylinder.	INTRO
+10	18	inactive	protein_state	While the inactive α subunits build the two outer rings, the β subunits form the inner rings.	INTRO
+19	29	α subunits	protein	While the inactive α subunits build the two outer rings, the β subunits form the inner rings.	INTRO
+50	55	rings	structure_element	While the inactive α subunits build the two outer rings, the β subunits form the inner rings.	INTRO
+61	71	β subunits	protein	While the inactive α subunits build the two outer rings, the β subunits form the inner rings.	INTRO
+87	92	rings	structure_element	While the inactive α subunits build the two outer rings, the β subunits form the inner rings.	INTRO
+38	48	β subunits	protein	Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.	INTRO
+57	59	β1	protein	Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.	INTRO
+61	63	β2	protein	Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.	INTRO
+68	70	β5	protein	Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.	INTRO
+88	114	proteolytic active centres	site	Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.	INTRO
+127	129	CP	complex_assembly	Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.	INTRO
+164	175	propeptides	structure_element	Only three out of the seven different β subunits, namely β1, β2 and β5, bear N-terminal proteolytic active centres, and before CP maturation these are protected by propeptides.	INTRO
+21	23	CP	complex_assembly	In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).	INTRO
+40	51	prosegments	structure_element	In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).	INTRO
+56	81	autocatalytically removed	ptm	In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).	INTRO
+117	136	active site residue	site	In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).	INTRO
+137	141	Thr1	residue_name_number	In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).	INTRO
+182	189	Gly(-1)	residue_name_number	In the last stage of CP biogenesis, the prosegments are autocatalytically removed through nucleophilic attack by the active site residue Thr1 on the preceding peptide bond involving Gly(-1).	INTRO
+15	26	propeptides	structure_element	Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides.	INTRO
+50	56	active	protein_state	Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides.	INTRO
+57	59	CP	complex_assembly	Release of the propeptides creates a functionally active CP that cleaves proteins into short peptides.	INTRO
+36	61	substrate-binding channel	site	Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds.	INTRO
+129	135	active	protein_state	Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds.	INTRO
+136	146	β subunits	protein	Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds.	INTRO
+152	164	active sites	site	Although the chemical nature of the substrate-binding channel and hence substrate preferences are unique to each of the distinct active β subunits, all active sites employ an identical reaction mechanism to hydrolyse peptide bonds.	INTRO
+23	27	Thr1	residue_name_number	Nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of the scissile peptide bond creates a first cleavage product and a covalent acyl-enzyme intermediate.	INTRO
+19	26	complex	complex_assembly	Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment.	INTRO
+61	66	water	chemical	Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment.	INTRO
+92	98	enzyme	complex_assembly	Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment.	INTRO
+123	130	peptide	chemical	Hydrolysis of this complex by the addition of a nucleophilic water molecule regenerates the enzyme and releases the second peptide fragment.	INTRO
+4	14	proteasome	complex_assembly	The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage.	INTRO
+40	80	N-terminal nucleophilic (Ntn) hydrolases	protein_type	The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage.	INTRO
+90	94	free	protein_state	The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage.	INTRO
+121	125	Thr1	residue_name_number	The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage.	INTRO
+158	162	Thr1	residue_name_number	The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage.	INTRO
+205	209	Thr1	residue_name_number	The proteasome belongs to the family of N-terminal nucleophilic (Ntn) hydrolases, and the free N-terminal amine group of Thr1 was proposed to deprotonate the Thr1 hydroxyl group to generate a nucleophilic Thr1Oγ for peptide-bond cleavage.	INTRO
+40	74	autocatalytic precursor processing	ptm	This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed.	INTRO
+90	98	immature	protein_state	This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed.	INTRO
+99	111	active sites	site	This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed.	INTRO
+113	117	Thr1	residue_name_number	This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed.	INTRO
+152	159	Gly(-1)	residue_name_number	This mechanism, however, cannot explain autocatalytic precursor processing because in the immature active sites, Thr1N is part of the peptide bond with Gly(-1), the bond that needs to be hydrolysed.	INTRO
+47	51	Thr1	residue_name_number	An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å).	INTRO
+88	93	Lys33	residue_name_number	An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å).	INTRO
+139	143	Thr1	residue_name_number	An alternative candidate for deprotonating the Thr1 hydroxyl group is the side chain of Lys33 as it is within hydrogen-bonding distance to Thr1OH (2.7 Å).	INTRO
+63	84	autocatalytic removal	ptm	In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage.	INTRO
+92	102	propeptide	structure_element	In principle it could function as the general base during both autocatalytic removal of the propeptide and protein substrate cleavage.	INTRO
+69	79	proteasome	complex_assembly	Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism.	INTRO
+80	91	active-site	site	Here we provide experimental evidences for this distinct view of the proteasome active-site mechanism.	INTRO
+10	45	biochemical and structural analyses	experimental_method	Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.	INTRO
+91	93	β5	protein	Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.	INTRO
+94	104	propeptide	structure_element	Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.	INTRO
+113	124	active site	site	Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.	INTRO
+228	252	autocatalytic activation	ptm	Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.	INTRO
+260	270	proteasome	complex_assembly	Data from biochemical and structural analyses of proteasome variants with mutations in the β5 propeptide and the active site strongly support the model and deliver novel insights into the structural constraints required for the autocatalytic activation of the proteasome.	INTRO
+44	47	Thr	residue_name	Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.	INTRO
+53	56	Cys	residue_name	Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.	INTRO
+60	63	Ser	residue_name	Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.	INTRO
+101	122	X-ray crystallography	experimental_method	Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.	INTRO
+137	167	activity and inhibition assays	experimental_method	Furthermore, we determine the advantages of Thr over Cys or Ser as the active-site nucleophile using X-ray crystallography together with activity and inhibition assays.	INTRO
+16	26	proteasome	complex_assembly	Inactivation of proteasome subunits by T1A mutations	RESULTS
+27	35	subunits	protein	Inactivation of proteasome subunits by T1A mutations	RESULTS
+39	42	T1A	mutant	Inactivation of proteasome subunits by T1A mutations	RESULTS
+43	52	mutations	experimental_method	Inactivation of proteasome subunits by T1A mutations	RESULTS
+0	10	Proteasome	complex_assembly	Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells.	RESULTS
+131	141	eukaryotic	taxonomy_domain	Proteasome-mediated degradation of cell-cycle regulators and potentially toxic misfolded proteins is required for the viability of eukaryotic cells.	RESULTS
+20	31	active site	site	Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.	RESULTS
+32	36	Thr1	residue_name_number	Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.	RESULTS
+40	51	mutation to	experimental_method	Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.	RESULTS
+52	55	Ala	residue_name	Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.	RESULTS
+126	136	proteasome	complex_assembly	Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.	RESULTS
+137	149	active sites	site	Inactivation of the active site Thr1 by mutation to Ala has been used to study substrate specificity and the hierarchy of the proteasome active sites.	RESULTS
+0	5	Yeast	taxonomy_domain	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+44	50	β1-T1A	mutant	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+54	60	β2-T1A	mutant	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+128	137	catalytic	protein_state	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+138	148	β subunits	protein	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+153	161	disabled	protein_state	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+166	183	carry remnants of	protein_state	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+201	212	propeptides	structure_element	Yeast strains carrying the single mutations β1-T1A or β2-T1A, or both, are viable, even though one or two of the three distinct catalytic β subunits are disabled and carry remnants of their N-terminal propeptides (Table 1).	RESULTS
+32	34	β1	protein	These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival.	RESULTS
+39	41	β2	protein	These results indicate that the β1 and β2 proteolytic activities are not essential for cell survival.	RESULTS
+17	20	T1A	mutant	By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so.	RESULTS
+41	43	β5	protein	By contrast, the T1A mutation in subunit β5 has been reported to be lethal or nearly so.	RESULTS
+29	35	β5-T1A	mutant	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+52	62	propeptide	structure_element	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+64	66	pp	chemical	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+68	100	deleted but expressed separately	experimental_method	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+104	109	trans	protein_state	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+111	117	β5-T1A	mutant	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+118	120	pp	chemical	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+121	126	trans	protein_state	Viability is restored if the β5-T1A subunit has its propeptide (pp) deleted but expressed separately in trans (β5-T1A pp trans), although substantial phenotypic impairment remains (Table 1).	RESULTS
+12	37	crystallographic analysis	experimental_method	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+45	51	β5-T1A	mutant	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+52	54	pp	chemical	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+55	60	trans	protein_state	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+61	67	mutant	protein_state	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+90	98	mutation	experimental_method	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+138	159	catalytic active site	site	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+173	188	trans-expressed	experimental_method	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+189	191	β5	protein	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+192	202	propeptide	structure_element	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+206	215	not bound	protein_state	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+223	225	β5	protein	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+226	251	substrate-binding channel	site	Our present crystallographic analysis of the β5-T1A pp trans mutant demonstrates that the mutation per se does not structurally alter the catalytic active site and that the trans-expressed β5 propeptide is not bound in the β5 substrate-binding channel (Supplementary Fig. 1a).	RESULTS
+33	39	β5-T1A	mutant	The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy.	RESULTS
+40	46	mutant	protein_state	The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy.	RESULTS
+47	49	pp	chemical	The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy.	RESULTS
+50	53	cis	protein_state	The extremely weak growth of the β5-T1A mutant pp cis described by Chen and Hochstrasser compared with the inviability reported by Heinemeyer et al. prompted us to analyse this discrepancy.	RESULTS
+0	26	Sequencing of the plasmids	experimental_method	Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods).	RESULTS
+59	64	yeast	taxonomy_domain	Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods).	RESULTS
+88	113	site-directed mutagenesis	experimental_method	Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods).	RESULTS
+132	138	β5-T1A	mutant	Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods).	RESULTS
+139	145	mutant	protein_state	Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods).	RESULTS
+146	148	pp	chemical	Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods).	RESULTS
+149	152	cis	protein_state	Sequencing of the plasmids, testing them in both published yeast strain backgrounds and site-directed mutagenesis revealed that the β5-T1A mutant pp cis is viable, but suffers from a marked growth defect that requires extended incubation of 4–5 days for initial colony formation (Table 1 and Supplementary Methods).	RESULTS
+48	52	K81R	mutant	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+64	66	β5	protein	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+112	143	This single amino-acid exchange	experimental_method	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+162	171	interface	site	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+188	190	α4	protein	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+192	194	β4	protein	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+199	201	β5	protein	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+251	253	CP	complex_assembly	We also identified an additional point mutation K81R in subunit β5 that was present in the allele used in ref.. This single amino-acid exchange is located at the interface of the subunits α4, β4 and β5 (Supplementary Fig. 1b) and might weakly promote CP assembly by enhancing inter-subunit contacts.	RESULTS
+34	45	β5-T1A-K81R	mutant	The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).	RESULTS
+46	52	mutant	protein_state	The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).	RESULTS
+77	94	crystal structure	evidence	The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).	RESULTS
+100	105	yeast	taxonomy_domain	The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).	RESULTS
+106	116	proteasome	complex_assembly	The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).	RESULTS
+118	121	yCP	complex_assembly	The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).	RESULTS
+132	138	β5-T1A	mutant	The slightly better growth of the β5-T1A-K81R mutant allowed us to solve the crystal structure of a yeast proteasome (yCP) with the β5-T1A mutation, which is discussed in the following section (for details see Supplementary Note 1).	RESULTS
+0	10	Propeptide	structure_element	Propeptide conformation and triggering of autolysis	RESULTS
+42	51	autolysis	ptm	Propeptide conformation and triggering of autolysis	RESULTS
+22	32	proteasome	complex_assembly	In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains.	RESULTS
+49	60	propeptides	structure_element	In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains.	RESULTS
+65	90	autocatalytically cleaved	ptm	In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains.	RESULTS
+100	106	mature	protein_state	In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains.	RESULTS
+107	124	β-subunit domains	protein	In the final steps of proteasome biogenesis, the propeptides are autocatalytically cleaved from the mature β-subunit domains.	RESULTS
+12	14	β1	protein	For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).	RESULTS
+73	83	propeptide	structure_element	For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).	RESULTS
+104	108	(-2)	residue_number	For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).	RESULTS
+147	168	S1 specificity pocket	site	For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).	RESULTS
+176	201	substrate-binding channel	site	For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).	RESULTS
+223	225	45	residue_number	For subunit β1, this process was previously inferred to require that the propeptide residue at position (-2) of the subunit precursor occupies the S1 specificity pocket of the substrate-binding channel formed by amino acid 45 (for details see Supplementary Note 2).	RESULTS
+38	48	prosegment	structure_element	Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).	RESULTS
+58	78	antiparallel β-sheet	structure_element	Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).	RESULTS
+86	97	active site	site	Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).	RESULTS
+108	115	Gly(-1)	residue_name_number	Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).	RESULTS
+125	144	γ-turn conformation	structure_element	Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).	RESULTS
+210	217	Leu(-2)	residue_name_number	Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).	RESULTS
+223	227	Thr1	residue_name_number	Furthermore, it was observed that the prosegment forms an antiparallel β-sheet in the active site, and that Gly(-1) adopts a γ-turn conformation, which by definition is characterized by a hydrogen bond between Leu(-2)O and Thr1NH (ref.).	RESULTS
+27	33	β1-T1A	mutant	Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).	RESULTS
+34	40	mutant	protein_state	Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).	RESULTS
+41	61	crystallographically	experimental_method	Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).	RESULTS
+93	103	structures	evidence	Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).	RESULTS
+111	117	β2-T1A	mutant	Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).	RESULTS
+129	142	β1-T1A-β2-T1A	mutant	Here we again analysed the β1-T1A mutant crystallographically but in addition determined the structures of the β2-T1A single and β1-T1A-β2-T1A double mutants (Protein Data Bank (PDB) entry codes are provided in Supplementary Table 1).	RESULTS
+11	13	β1	protein	In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a).	RESULTS
+29	36	Gly(-1)	residue_name_number	In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a).	RESULTS
+52	62	sharp turn	structure_element	In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a).	RESULTS
+81	100	prosegment cleavage	ptm	In subunit β1, we found that Gly(-1) indeed forms a sharp turn, which relaxes on prosegment cleavage (Fig. 1a and Supplementary Fig. 2a).	RESULTS
+13	32	γ-turn conformation	structure_element	However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1.	RESULTS
+198	205	Gly(-1)	residue_name_number	However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1.	RESULTS
+233	237	Thr1	residue_name_number	However, the γ-turn conformation and the associated hydrogen bond initially proposed is for geometric and chemical reasons inappropriate and would not perfectly position the carbonyl carbon atom of Gly(-1) for nucleophilic attack by Thr1.	RESULTS
+14	16	β2	protein	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+17	27	propeptide	structure_element	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+29	36	Thr(-2)	residue_name_number	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+50	59	S1 pocket	site	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+102	109	Leu(-2)	residue_name_number	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+113	115	β1	protein	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+156	158	β2	protein	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+159	168	S1 pocket	site	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+180	185	Gly45	residue_name_number	Regarding the β2 propeptide, Thr(-2) occupies the S1 pocket but is less deeply anchored compared with Leu(-2) in β1, which might be due to the rather large β2-S1 pocket created by Gly45.	RESULTS
+0	7	Thr(-2)	residue_name_number	Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b).	RESULTS
+18	25	Gly(-1)	residue_name_number	Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b).	RESULTS
+112	116	Thr1	residue_name_number	Thr(-2) positions Gly(-1)O via hydrogen bonding (∼2.8 Å) in a perfect trajectory for the nucleophilic attack by Thr1Oγ (Fig. 1b and Supplementary Fig. 2b).	RESULTS
+38	40	β5	protein	Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant.	RESULTS
+41	51	propeptide	structure_element	Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant.	RESULTS
+59	70	β5-T1A-K81R	mutant	Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant.	RESULTS
+71	77	mutant	protein_state	Next, we examined the position of the β5 propeptide in the β5-T1A-K81R mutant.	RESULTS
+14	21	Gly(-1)	residue_name_number	Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet.	RESULTS
+60	69	histidine	residue_name	Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet.	RESULTS
+93	97	(-2)	residue_number	Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet.	RESULTS
+112	114	S2	site	Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet.	RESULTS
+130	139	S1 pocket	site	Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet.	RESULTS
+164	184	antiparallel β-sheet	structure_element	Surprisingly, Gly(-1) is completely extended and forces the histidine side chain at position (-2) to occupy the S2 instead of the S1 pocket, thereby disrupting the antiparallel β-sheet.	RESULTS
+36	43	Gly(-1)	residue_name_number	Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d).	RESULTS
+95	99	Thr1	residue_name_number	Nonetheless, the carbonyl carbon of Gly(-1) would be ideally placed for nucleophilic attack by Thr1Oγ (Fig. 1c and Supplementary Fig. 2c,d).	RESULTS
+7	11	K81R	mutant	As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded.	RESULTS
+45	56	active site	site	As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded.	RESULTS
+58	62	Thr1	residue_name_number	As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded.	RESULTS
+65	70	Arg81	residue_name_number	As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded.	RESULTS
+98	108	propeptide	structure_element	As the K81R mutation is located far from the active site (Thr1Cα–Arg81Cα: 24 Å), any influence on propeptide conformation can be excluded.	RESULTS
+31	33	β5	protein	Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.	RESULTS
+34	43	S1 pocket	site	Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.	RESULTS
+84	89	Met45	residue_name_number	Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.	RESULTS
+128	135	His(-2)	residue_name_number	Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.	RESULTS
+143	150	S1 site	site	Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.	RESULTS
+201	210	autolysis	ptm	Instead, the plasticity of the β5 S1 pocket caused by the rotational flexibility of Met45 might prevent stable accommodation of His(-2) in the S1 site and thus also promote its immediate release after autolysis.	RESULTS
+61	65	Thr1	residue_name_number	Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown.	RESULTS
+106	115	autolysis	ptm	Processing of β-subunit precursors requires deprotonation of Thr1OH; however, the general base initiating autolysis is unknown.	RESULTS
+12	22	eukaryotic	taxonomy_domain	Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a).	RESULTS
+35	37	β5	protein	Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a).	RESULTS
+54	57	His	residue_name	Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a).	RESULTS
+78	82	(-2)	residue_number	Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a).	RESULTS
+90	100	propeptide	structure_element	Remarkably, eukaryotic proteasomal β5 subunits bear a His residue in position (-2) of the propeptide (Supplementary Fig. 3a).	RESULTS
+3	12	histidine	residue_name	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+59	75	catalytic triads	site	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+79	95	serine proteases	protein_type	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+125	132	His(-2)	residue_name_number	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+154	156	β5	protein	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+157	167	propeptide	structure_element	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+171	188	exchanging it for	experimental_method	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+189	192	Asn	residue_name	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+194	197	Lys	residue_name	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+199	202	Phe	residue_name	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+207	210	Ala	residue_name	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+216	221	yeast	taxonomy_domain	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+303	309	H(-2)N	mutant	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+314	320	H(-2)F	mutant	As histidine commonly functions as a proton shuttle in the catalytic triads of serine proteases, we investigated the role of His(-2) in processing of the β5 propeptide by exchanging it for Asn, Lys, Phe and Ala. All yeast mutants were viable at 30 °C, but suffered from growth defects at 37 °C with the H(-2)N and H(-2)F mutants being most affected (Supplementary Fig. 3b and Table 1).	RESULTS
+56	62	H(-2)N	mutant	In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c).	RESULTS
+67	73	H(-2)F	mutant	In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c).	RESULTS
+74	80	mutant	protein_state	In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c).	RESULTS
+81	85	yCPs	complex_assembly	In agreement, the chymotrypsin-like (ChT-L) activity of H(-2)N and H(-2)F mutant yCPs was impaired in situ and in vitro (Supplementary Fig. 3c).	RESULTS
+0	19	Structural analyses	experimental_method	Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities.	RESULTS
+38	49	propeptides	structure_element	Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities.	RESULTS
+57	63	mutant	protein_state	Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities.	RESULTS
+64	68	yCPs	complex_assembly	Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities.	RESULTS
+85	110	2FO–FC electron densities	evidence	Structural analyses revealed that the propeptides of all mutant yCPs shared residual 2FO–FC electron densities.	RESULTS
+0	7	Gly(-1)	residue_name_number	Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned.	RESULTS
+12	15	Phe	residue_name	Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned.	RESULTS
+16	23	Lys(-2)	residue_name_number	Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned.	RESULTS
+64	67	Ala	residue_name	Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned.	RESULTS
+68	75	Asn(-2)	residue_name_number	Gly(-1) and Phe/Lys(-2) were visualized at low occupancy, while Ala/Asn(-2) could not be assigned.	RESULTS
+40	49	processed	protein_state	This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.	RESULTS
+54	65	unprocessed	protein_state	This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.	RESULTS
+66	68	β5	protein	This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.	RESULTS
+101	110	autolysis	ptm	This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.	RESULTS
+160	164	(-2)	residue_number	This observation indicates a mixture of processed and unprocessed β5 subunits and partially impaired autolysis, thereby excluding any essential role of residue (-2) as the general base.	RESULTS
+40	44	(-2)	residue_number	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+71	81	propeptide	structure_element	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+85	114	creating mutants that combine	experimental_method	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+119	122	T1A	mutant	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+124	128	K81R	mutant	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+130	141	mutation(s)	experimental_method	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+147	153	H(-2)L	mutant	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+155	161	H(-2)T	mutant	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+165	171	H(-2)A	mutant	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+172	185	substitutions	experimental_method	Next, we examined the effect of residue (-2) on the orientation of the propeptide by creating mutants that combine the T1A (K81R) mutation(s) with H(-2)L, H(-2)T or H(-2)A substitutions.	RESULTS
+0	7	Leu(-2)	residue_name_number	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+26	31	yeast	taxonomy_domain	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+32	34	β1	protein	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+78	85	Thr(-2)	residue_name_number	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+107	109	β2	protein	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+110	121	propeptides	structure_element	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+151	158	Ala(-2)	residue_name_number	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+183	185	β5	protein	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+186	195	S1 pocket	site	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+239	244	Met45	residue_name_number	Leu(-2) is encoded in the yeast β1 subunit precursor (Supplementary Fig. 3a); Thr(-2) is generally part of β2-propeptides (Supplementary Fig. 3a); and Ala(-2) was expected to fit the β5-S1 pocket without inducing conformational changes of Met45, allowing it to accommodate ‘β1-like' propeptide positioning.	RESULTS
+17	23	β5-T1A	mutant	As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1).	RESULTS
+37	43	yeasts	taxonomy_domain	As expected from β5-T1A mutants, the yeasts show severe growth phenotypes, with minor variations (Supplementary Fig. 4a and Table 1).	RESULTS
+14	32	crystal structures	evidence	We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1).	RESULTS
+40	53	β5-H(-2)L-T1A	mutant	We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1).	RESULTS
+55	68	β5-H(-2)T-T1A	mutant	We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1).	RESULTS
+77	95	β5-H(-2)A-T1A-K81R	mutant	We determined crystal structures of the β5-H(-2)L-T1A, β5-H(-2)T-T1A and the β5-H(-2)A-T1A-K81R mutants (Supplementary Table 1).	RESULTS
+8	26	β5-H(-2)A-T1A-K81R	mutant	For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).	RESULTS
+54	61	Gly(-1)	residue_name_number	For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).	RESULTS
+66	73	Ala(-2)	residue_name_number	For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).	RESULTS
+111	118	Ala(-2)	residue_name_number	For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).	RESULTS
+162	172	propeptide	structure_element	For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).	RESULTS
+180	205	substrate-binding channel	site	For the β5-H(-2)A-T1A-K81R variant, only the residues Gly(-1) and Ala(-2) could be visualized, indicating that Ala(-2) leads to insufficient stabilization of the propeptide in the substrate-binding channel (Supplementary Fig. 4d).	RESULTS
+17	28	prosegments	structure_element	By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.	RESULTS
+36	49	β5-H(-2)L-T1A	mutant	By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.	RESULTS
+58	71	β5-H(-2)T-T1A	mutant	By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.	RESULTS
+122	150	2FO–FC electron-density maps	evidence	By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.	RESULTS
+258	268	propeptide	structure_element	By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.	RESULTS
+277	284	His(-2)	residue_name_number	By contrast, the prosegments of the β5-H(-2)L-T1A and the β5-H(-2)T-T1A mutants were significantly better resolved in the 2FO–FC electron-density maps yet not at full occupancy (Supplementary Fig. 4b,c and Supplementary Table 1), suggesting that the natural propeptide bearing His(-2) is most favourable.	RESULTS
+19	26	Leu(-2)	residue_name_number	Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h).	RESULTS
+31	38	Thr(-2)	residue_name_number	Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h).	RESULTS
+64	85	S1 specificity pocket	site	Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h).	RESULTS
+96	101	Met45	residue_name_number	Nevertheless, both Leu(-2) and Thr(-2) were found to occupy the S1 specificity pocket formed by Met45 (Fig. 2a,b and Supplementary Fig. 4f–h).	RESULTS
+48	55	His(-2)	residue_name_number	This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site.	RESULTS
+63	65	β5	protein	This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site.	RESULTS
+66	76	propeptide	structure_element	This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site.	RESULTS
+106	113	S1 site	site	This result proves that the naturally occurring His(-2) of the β5 propeptide does not stably fit into the S1 site.	RESULTS
+6	13	Gly(-1)	residue_name_number	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+47	56	wild-type	protein_state	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+58	60	WT	protein_state	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+66	72	mutant	protein_state	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+73	75	β5	protein	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+76	87	propeptides	structure_element	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+179	183	Thr1	residue_name_number	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+240	244	(-2)	residue_number	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+252	261	S1 pocket	site	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+283	303	antiparallel β-sheet	structure_element	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+321	330	autolysis	ptm	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+338	348	propeptide	structure_element	Since Gly(-1) adopts the same position in both wild-type (WT) and mutant β5 propeptides, and since in all cases its carbonyl carbon is perfectly placed for nucleophilic attack by Thr1Oγ (Fig. 2b), we propose that neither binding of residue (-2) to the S1 pocket nor formation of the antiparallel β-sheet is essential for autolysis of the propeptide.	RESULTS
+24	41	crystal structure	evidence	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+47	55	chimeric	protein_state	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+56	59	yCP	complex_assembly	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+71	76	yeast	taxonomy_domain	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+77	79	β1	protein	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+80	90	propeptide	structure_element	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+91	102	replaced by	experimental_method	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+107	109	β5	protein	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+110	121	counterpart	structure_element	Next, we determined the crystal structure of a chimeric yCP having the yeast β1-propeptide replaced by its β5 counterpart.	RESULTS
+34	57	2FO–FC electron density	evidence	Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable.	RESULTS
+65	67	β1	protein	Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable.	RESULTS
+68	79	active site	site	Although we observed fragments of 2FO–FC electron density in the β1 active site, the data were not interpretable.	RESULTS
+36	43	Thr(-2)	residue_name_number	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+47	49	β2	protein	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+51	58	Leu(-2)	residue_name_number	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+70	72	β1	protein	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+76	89	not conserved	protein_state	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+132	139	created	experimental_method	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+142	151	β2-T(-2)V	mutant	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+152	162	proteasome	complex_assembly	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+163	169	mutant	protein_state	Bearing in mind that in contrast to Thr(-2) in β2, Leu(-2) in subunit β1 is not conserved among species (Supplementary Fig. 3a), we created a β2-T(-2)V proteasome mutant.	RESULTS
+17	23	β2-T1A	mutant	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+24	42	crystal structures	evidence	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+44	51	Thr(-2)	residue_name_number	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+70	77	Gly(-1)	residue_name_number	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+131	144	β5-H(-2)T-T1A	mutant	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+145	151	mutant	protein_state	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+191	199	exchange	experimental_method	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+203	210	Thr(-2)	residue_name_number	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+214	217	Val	residue_name	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+221	223	β2	protein	As proven by the β2-T1A crystal structures, Thr(-2) hydrogen bonds to Gly(-1)O. Although this interaction was not observed for the β5-H(-2)T-T1A mutant (Fig. 2c and Supplementary Fig. 4c,i), exchange of Thr(-2) by Val in β2, a conservative mutation regarding size but drastic with respect to polarity, was found to inhibit maturation of this subunit (Fig. 2d and Supplementary Fig. 4e,j).	RESULTS
+13	40	2FO–FC electron-density map	evidence	Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome.	RESULTS
+82	84	β2	protein	Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome.	RESULTS
+85	95	propeptide	structure_element	Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome.	RESULTS
+127	133	β2-T1A	mutant	Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome.	RESULTS
+134	144	proteasome	complex_assembly	Notably, the 2FO–FC electron-density map displays a different orientation for the β2 propeptide than has been observed for the β2-T1A proteasome.	RESULTS
+15	22	Val(-2)	residue_name_number	In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k).	RESULTS
+45	52	S1 site	site	In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k).	RESULTS
+57	64	Gly(-1)	residue_name_number	In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k).	RESULTS
+176	180	Thr1	residue_name_number	In particular, Val(-2) is displaced from the S1 site and Gly(-1) is severely shifted (movement of the carbonyl oxygen atom of 3.8 Å), thereby preventing nucleophilic attack of Thr1 (Fig. 2d and Supplementary Fig. 4j,k).	RESULTS
+62	82	active-site residues	site	These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome.	RESULTS
+87	94	Gly(-1)	residue_name_number	These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome.	RESULTS
+133	143	proteasome	complex_assembly	These results further confirm that correct positioning of the active-site residues and Gly(-1) is decisive for the maturation of the proteasome.	RESULTS
+4	15	active site	site	The active site of the proteasome	RESULTS
+23	33	proteasome	complex_assembly	The active site of the proteasome	RESULTS
+38	49	active site	site	Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis.	RESULTS
+50	54	Thr1	residue_name_number	Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis.	RESULTS
+60	64	Thr1	residue_name_number	Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis.	RESULTS
+87	92	water	chemical	Proton shuttling from the proteasomal active site Thr1OH to Thr1NH2 via a nucleophilic water molecule was suggested to initiate peptide-bond hydrolysis.	RESULTS
+16	24	immature	protein_state	However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ.	RESULTS
+25	33	particle	complex_assembly	However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ.	RESULTS
+34	38	Thr1	residue_name_number	However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ.	RESULTS
+60	70	propeptide	structure_element	However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ.	RESULTS
+91	95	Thr1	residue_name_number	However, in the immature particle Thr1NH2 is blocked by the propeptide and cannot activate Thr1Oγ.	RESULTS
+9	14	Lys33	residue_name_number	Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor.	RESULTS
+60	64	Thr1	residue_name_number	Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor.	RESULTS
+82	102	catalytically active	protein_state	Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor.	RESULTS
+103	113	β subunits	protein	Instead, Lys33NH2, which is in hydrogen-bonding distance to Thr1Oγ (2.7 Å) in all catalytically active β subunits (Fig. 3a,b), was proposed to serve as the proton acceptor.	RESULTS
+11	27	catalytic tetrad	site	A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.	RESULTS
+44	48	Thr1	residue_name_number	A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.	RESULTS
+52	56	Thr1	residue_name_number	A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.	RESULTS
+61	66	Lys33	residue_name_number	A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.	RESULTS
+74	79	Asp17	residue_name_number	A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.	RESULTS
+109	114	water	chemical	A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.	RESULTS
+208	219	active site	site	A proposed catalytic tetrad model involving Thr1OH, Thr1NH2, Lys33NH2 and Asp17Oδ, as well as a nucleophilic water molecule as the proton shuttle appeared to accommodate all possible views of the proteasomal active site.	RESULTS
+39	42	yCP	complex_assembly	Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base.	RESULTS
+43	59	X-ray structures	evidence	Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base.	RESULTS
+98	109	active site	site	Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base.	RESULTS
+117	127	proteasome	complex_assembly	Twenty years later, with a plethora of yCP X-ray structures in hand, we decided to re-analyse the active site of the proteasome and to resolve the uncertainty regarding the nature of the general base.	RESULTS
+0	8	Mutation	experimental_method	Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).	RESULTS
+12	14	β5	protein	Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).	RESULTS
+15	20	Lys33	residue_name_number	Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).	RESULTS
+24	27	Ala	residue_name	Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).	RESULTS
+82	117	structural and biochemical analyses	experimental_method	Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).	RESULTS
+162	181	propeptide cleavage	ptm	Mutation of β5-Lys33 to Ala causes a strongly deleterious phenotype, and previous structural and biochemical analyses confirmed that this is caused by failure of propeptide cleavage, and consequently, lack of ChT-L activity (Fig. 4a, Supplementary Fig. 3b and Table 1; for details see Supplementary Note 1).	RESULTS
+21	28	β5-K33A	mutant	The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a).	RESULTS
+29	35	mutant	protein_state	The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a).	RESULTS
+77	88	β5-T1A-K81R	mutant	The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a).	RESULTS
+89	94	yeast	taxonomy_domain	The phenotype of the β5-K33A mutant was however less pronounced than for the β5-T1A-K81R yeast (Fig. 4a).	RESULTS
+70	77	L(-49)S	mutant	This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1).	RESULTS
+85	87	β5	protein	This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1).	RESULTS
+88	98	propeptide	structure_element	This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1).	RESULTS
+106	113	β5-K33A	mutant	This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1).	RESULTS
+114	120	mutant	protein_state	This discrepancy in growth was traced to an additional point mutation L(-49)S in the β5-propeptide of the β5-K33A mutant (see also Supplementary Note 1).	RESULTS
+0	21	Structural comparison	experimental_method	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+29	44	β5-L(-49)S-K33A	mutant	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+49	60	β5-T1A-K81R	mutant	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+61	73	active sites	site	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+88	96	mutation	experimental_method	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+100	105	Lys33	residue_name_number	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+109	112	Ala	residue_name	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+150	154	Thr1	residue_name_number	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+171	181	propeptide	structure_element	Structural comparison of the β5-L(-49)S-K33A and β5-T1A-K81R active sites revealed that mutation of Lys33 to Ala creates a cavity that is filled with Thr1 and the remnant propeptide.	RESULTS
+36	47	active-site	site	This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a).	RESULTS
+98	100	β5	protein	This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a).	RESULTS
+101	112	active site	site	This structural alteration destroys active-site integrity and abolishes catalytic activity of the β5 active site (Supplementary Fig. 5a).	RESULTS
+41	46	Lys33	residue_name_number	Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).	RESULTS
+69	76	β5-K33A	mutant	Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).	RESULTS
+77	83	mutant	protein_state	Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).	RESULTS
+94	104	propeptide	structure_element	Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).	RESULTS
+105	125	expressed separately	experimental_method	Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).	RESULTS
+149	151	pp	chemical	Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).	RESULTS
+152	157	trans	protein_state	Additional proof for the key function of Lys33 was obtained from the β5-K33A mutant, with the propeptide expressed separately from the main subunit (pp trans).	RESULTS
+4	8	Thr1	residue_name_number	The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b).	RESULTS
+28	34	mutant	protein_state	The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b).	RESULTS
+57	67	propeptide	structure_element	The Thr1 N terminus of this mutant is not blocked by the propeptide, yet its catalytic activity is reduced by ∼83% (Supplementary Fig. 6b).	RESULTS
+26	43	crystal structure	evidence	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+51	58	β5-K33A	mutant	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+59	61	pp	chemical	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+62	67	trans	protein_state	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+68	74	mutant	protein_state	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+75	90	in complex with	protein_state	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+91	102	carfilzomib	chemical	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+154	156	β5	protein	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+157	169	active sites	site	Consistent with this, the crystal structure of the β5-K33A pp trans mutant in complex with carfilzomib only showed partial occupancy of the ligand at the β5 active sites (Supplementary Fig. 5b and Supplementary Table 1).	RESULTS
+9	20	acetylation	ptm	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+28	32	Thr1	residue_name_number	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+65	72	β5-K33A	mutant	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+73	75	pp	chemical	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+76	81	trans	protein_state	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+82	85	apo	protein_state	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+86	103	crystal structure	evidence	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+177	182	Lys33	residue_name_number	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+199	203	Thr1	residue_name_number	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+235	239	Thr1	residue_name_number	Since no acetylation of the Thr1 N terminus was observed for the β5-K33A pp trans apo crystal structure, the reduced reactivity towards substrates and inhibitors indicates that Lys33NH2, rather than Thr1NH2, deprotonates and activates Thr1OH.	RESULTS
+17	34	crystal structure	evidence	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+42	49	β5-K33A	mutant	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+50	52	pp	chemical	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+53	58	trans	protein_state	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+59	65	mutant	protein_state	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+66	83	without inhibitor	protein_state	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+98	102	Thr1	residue_name_number	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+141	146	water	chemical	Furthermore, the crystal structure of the β5-K33A pp trans mutant without inhibitor revealed that Thr1Oγ strongly coordinates a well-defined water molecule (∼2 Å; Fig. 3c and Supplementary Fig. 5c,d).	RESULTS
+5	10	water	chemical	This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant.	RESULTS
+34	39	Arg19	residue_name_number	This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant.	RESULTS
+54	59	Asp17	residue_name_number	This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant.	RESULTS
+128	134	mutant	protein_state	This water hydrogen bonds also to Arg19O (∼3.0 Å) and Asp17Oδ (∼3.0 Å), and thereby presumably enables residual activity of the mutant.	RESULTS
+73	78	Lys33	residue_name_number	Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.	RESULTS
+89	91	WT	protein_state	Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.	RESULTS
+92	102	proteasome	complex_assembly	Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.	RESULTS
+103	112	structure	evidence	Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.	RESULTS
+168	173	Lys33	residue_name_number	Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.	RESULTS
+198	207	autolysis	ptm	Remarkably, the solvent molecule occupies the position normally taken by Lys33NH2 in the WT proteasome structure (Fig. 3c), further corroborating the essential role of Lys33 as the general base for autolysis and proteolysis.	RESULTS
+0	25	Conservative substitution	experimental_method	Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1).	RESULTS
+29	34	Lys33	residue_name_number	Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1).	RESULTS
+38	41	Arg	residue_name	Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1).	RESULTS
+49	58	autolysis	ptm	Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1).	RESULTS
+66	68	β5	protein	Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1).	RESULTS
+91	96	yeast	taxonomy_domain	Conservative substitution of Lys33 by Arg delays autolysis of the β5 precursor and impairs yeast growth (for details see Supplementary Note 1).	RESULTS
+6	10	Thr1	residue_name_number	While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).	RESULTS
+44	46	WT	protein_state	While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).	RESULTS
+47	51	yCPs	complex_assembly	While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).	RESULTS
+53	58	Arg33	residue_name_number	While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).	RESULTS
+89	94	Asp17	residue_name_number	While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).	RESULTS
+121	123	β5	protein	While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).	RESULTS
+124	135	active site	site	While Thr1 occupies the same position as in WT yCPs, Arg33 is unable to hydrogen bond to Asp17, thereby inactivating the β5 active site (Supplementary Fig. 5e).	RESULTS
+4	25	conservative mutation	experimental_method	The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1).	RESULTS
+29	34	Asp17	residue_name_number	The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1).	RESULTS
+38	41	Asn	residue_name	The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1).	RESULTS
+53	55	β5	protein	The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1).	RESULTS
+63	66	yCP	complex_assembly	The conservative mutation of Asp17 to Asn in subunit β5 of the yCP also provokes a severe growth defect (Supplementary Note 1, Supplementary Fig. 6a and Table 1).	RESULTS
+49	56	L(-49)S	mutant	Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP.	RESULTS
+72	74	β5	protein	Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP.	RESULTS
+75	85	propeptide	structure_element	Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP.	RESULTS
+124	131	β5-D17N	mutant	Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP.	RESULTS
+132	138	mutant	protein_state	Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP.	RESULTS
+139	142	yCP	complex_assembly	Notably, only with the additional point mutation L(-49)S present in the β5 propeptide could we purify a small amount of the β5-D17N mutant yCP.	RESULTS
+17	42	crystallographic analysis	experimental_method	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+49	55	mutant	protein_state	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+56	58	β5	protein	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+71	90	partially processed	protein_state	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+147	157	proteasome	complex_assembly	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+168	179	carfilzomib	chemical	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+207	209	β1	protein	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+214	216	β2	protein	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+227	229	WT	protein_state	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+230	232	β5	protein	As determined by crystallographic analysis, this mutant β5 subunit was partially processed (Table 1) but displayed impaired reactivity towards the proteasome inhibitor carfilzomib compared with the subunits β1 and β2, and with WT β5 (Supplementary Fig. 7a).	RESULTS
+19	22	cis	protein_state	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+34	44	expression	experimental_method	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+52	54	β5	protein	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+55	65	propeptide	structure_element	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+69	74	trans	protein_state	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+99	108	isolation	experimental_method	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+113	128	crystallization	experimental_method	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+136	140	D17N	mutant	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+141	147	mutant	protein_state	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+148	158	proteasome	complex_assembly	In contrast to the cis-construct, expression of the β5 propeptide in trans allowed straightforward isolation and crystallization of the D17N mutant proteasome.	RESULTS
+26	33	β5-D17N	mutant	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+34	36	pp	chemical	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+40	45	trans	protein_state	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+46	48	CP	complex_assembly	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+71	73	β5	protein	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+91	142	N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin	chemical	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+144	156	Suc-LLVY-AMC	chemical	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+162	205	carboxybenzyl-Gly-Gly-Leu-para-nitroanilide	chemical	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+207	216	Z-GGL-pNA	chemical	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+280	285	Asp17	residue_name_number	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+349	355	mature	protein_state	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+356	366	proteasome	complex_assembly	The ChT-L activity of the β5-D17N pp in trans CP towards the canonical β5 model substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) and carboxybenzyl-Gly-Gly-Leu-para-nitroanilide (Z-GGL-pNA) was severely reduced (Supplementary Fig. 6b), confirming that Asp17 is of fundamental importance for the catalytic activity of the mature proteasome.	RESULTS
+16	23	β5-D17N	mutant	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+24	26	pp	chemical	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+27	32	trans	protein_state	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+33	36	yCP	complex_assembly	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+37	54	crystal structure	evidence	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+81	83	WT	protein_state	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+84	87	yCP	complex_assembly	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+117	137	co-crystal structure	evidence	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+147	165	α′, β′ epoxyketone	chemical	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+176	187	carfilzomib	chemical	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+243	245	β5	protein	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+246	257	active site	site	Even though the β5-D17N pp trans yCP crystal structure appeared identical to the WT yCP (Supplementary Fig. 7b), the co-crystal structure with the α′, β′ epoxyketone inhibitor carfilzomib visualized only partial occupancy of the ligand in the β5 active site (Supplementary Fig. 7a).	RESULTS
+69	71	β5	protein	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+72	76	Thr1	residue_name_number	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+85	102	crystal structure	evidence	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+110	117	β5-D17N	mutant	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+118	120	pp	chemical	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+121	124	cis	protein_state	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+125	131	mutant	protein_state	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+132	147	in complex with	protein_state	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+148	159	carfilzomib	chemical	This observation is consistent with a strongly reduced reactivity of β5-Thr1 and the crystal structure of the β5-D17N pp cis mutant in complex with carfilzomib.	RESULTS
+0	9	Autolysis	ptm	Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1.	RESULTS
+49	56	β5-D17N	mutant	Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1.	RESULTS
+106	111	Asn17	residue_name_number	Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1.	RESULTS
+163	168	Lys33	residue_name_number	Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1.	RESULTS
+191	195	Thr1	residue_name_number	Autolysis and residual catalytic activity of the β5-D17N mutants may originate from the carbonyl group of Asn17, which albeit to a lower degree still can polarize Lys33 for the activation of Thr1.	RESULTS
+17	21	E17A	mutant	In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.	RESULTS
+22	28	mutant	protein_state	In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.	RESULTS
+48	57	β-subunit	protein	In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.	RESULTS
+65	73	archaeon	taxonomy_domain	In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.	RESULTS
+74	98	Thermoplasma acidophilum	species	In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.	RESULTS
+108	117	autolysis	ptm	In agreement, an E17A mutant in the proteasomal β-subunit of the archaeon Thermoplasma acidophilum prevents autolysis and catalysis.	RESULTS
+25	35	X-ray data	evidence	Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).	RESULTS
+43	50	β5-D17N	mutant	Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).	RESULTS
+51	57	mutant	protein_state	Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).	RESULTS
+67	77	propeptide	structure_element	Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).	RESULTS
+78	87	expressed	experimental_method	Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).	RESULTS
+91	94	cis	protein_state	Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).	RESULTS
+102	107	trans	protein_state	Strikingly, although the X-ray data on the β5-D17N mutant with the propeptide expressed in cis and in trans looked similar, there was a pronounced difference in their growth phenotypes observed (Supplementary Fig. 6a and Supplementary Fig. 7b).	RESULTS
+47	50	CPs	complex_assembly	On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).	RESULTS
+82	97	catalytic triad	site	On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).	RESULTS
+112	116	Thr1	residue_name_number	On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).	RESULTS
+118	123	Lys33	residue_name_number	On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).	RESULTS
+128	131	Asp	residue_name	On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).	RESULTS
+132	137	Glu17	residue_name_number	On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).	RESULTS
+147	181	autocatalytic precursor processing	ptm	On the basis of these results, we propose that CPs from all domains of life use a catalytic triad consisting of Thr1, Lys33 and Asp/Glu17 for both autocatalytic precursor processing and proteolysis (Fig. 3d).	RESULTS
+60	65	water	chemical	This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2.	RESULTS
+94	100	mature	protein_state	This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2.	RESULTS
+101	103	WT	protein_state	This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2.	RESULTS
+116	127	active site	site	This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2.	RESULTS
+163	167	Thr1	residue_name_number	This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2.	RESULTS
+173	177	Thr1	residue_name_number	This model is also consistent with the fact that no defined water molecule is observed in the mature WT proteasomal active site that could shuttle the proton from Thr1Oγ to Thr1NH2.	RESULTS
+16	27	active-site	site	To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit.	RESULTS
+46	69	exchanged the conserved	experimental_method	To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit.	RESULTS
+70	76	Asp166	residue_name_number	To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit.	RESULTS
+89	92	Asn	residue_name	To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit.	RESULTS
+100	105	yeast	taxonomy_domain	To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit.	RESULTS
+106	108	β5	protein	To explore this active-site model further, we exchanged the conserved Asp166 residue for Asn in the yeast β5 subunit.	RESULTS
+0	6	Asp166	residue_name_number	Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis.	RESULTS
+31	35	Thr1	residue_name_number	Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis.	RESULTS
+43	49	Ser129	residue_name_number	Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis.	RESULTS
+56	62	Ser169	residue_name_number	Asp166Oδ is hydrogen-bonded to Thr1NH2 via Ser129OH and Ser169OH, and therefore was proposed to be involved in catalysis.	RESULTS
+4	12	β5-D166N	mutant	The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1).	RESULTS
+13	15	pp	chemical	The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1).	RESULTS
+16	19	cis	protein_state	The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1).	RESULTS
+20	25	yeast	taxonomy_domain	The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1).	RESULTS
+26	32	mutant	protein_state	The β5-D166N pp cis yeast mutant is significantly impaired in growth and its ChT-L activity is drastically reduced (Supplementary Fig. 6a,b and Table 1).	RESULTS
+0	10	X-ray data	evidence	X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a).	RESULTS
+18	26	β5-D166N	mutant	X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a).	RESULTS
+27	33	mutant	protein_state	X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a).	RESULTS
+52	54	β5	protein	X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a).	RESULTS
+55	65	propeptide	structure_element	X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a).	RESULTS
+109	115	Ser129	residue_name_number	X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a).	RESULTS
+140	146	Asn166	residue_name_number	X-ray data on the β5-D166N mutant indicate that the β5 propeptide is hydrolysed, but due to reorientation of Ser129OH, the interaction with Asn166Oδ is disrupted (Supplementary Fig. 8a).	RESULTS
+11	16	water	chemical	Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing.	RESULTS
+29	37	bound to	protein_state	Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing.	RESULTS
+38	44	Ser129	residue_name_number	Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing.	RESULTS
+51	55	Thr1	residue_name_number	Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing.	RESULTS
+101	121	precursor processing	ptm	Instead, a water molecule is bound to Ser129OH and Thr1NH2 (Supplementary Fig. 8b), which may enable precursor processing.	RESULTS
+29	35	Ser169	residue_name_number	The hydrogen bonds involving Ser169OH are intact and may account for residual substrate turnover.	RESULTS
+0	7	Soaking	experimental_method	Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).	RESULTS
+12	20	β5-D166N	mutant	Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).	RESULTS
+21	29	crystals	experimental_method	Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).	RESULTS
+35	46	carfilzomib	chemical	Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).	RESULTS
+51	56	MG132	chemical	Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).	RESULTS
+94	98	Thr1	residue_name_number	Soaking the β5-D166N crystals with carfilzomib and MG132 resulted in covalent modification of Thr1 at high occupancy (Supplementary Fig. 8c).	RESULTS
+7	26	carfilzomib complex	complex_assembly	In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.	RESULTS
+27	36	structure	evidence	In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.	RESULTS
+38	42	Thr1	residue_name_number	In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.	RESULTS
+49	53	Thr1	residue_name_number	In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.	RESULTS
+104	110	Ser129	residue_name_number	In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.	RESULTS
+122	124	WT	protein_state	In the carfilzomib complex structure, Thr1Oγ and Thr1N incorporate into a morpholine ring structure and Ser129 adopts its WT-like orientation.	RESULTS
+7	24	MG132-bound state	protein_state	In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.	RESULTS
+26	30	Thr1	residue_name_number	In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.	RESULTS
+35	45	unmodified	protein_state	In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.	RESULTS
+73	79	Ser129	residue_name_number	In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.	RESULTS
+104	109	water	chemical	In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.	RESULTS
+130	136	Asn166	residue_name_number	In the MG132-bound state, Thr1N is unmodified, and we again observe that Ser129 is hydrogen-bonded to a water molecule instead of Asn166.	RESULTS
+8	11	Asn	residue_name	Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis.	RESULTS
+39	45	Asp166	residue_name_number	Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis.	RESULTS
+91	94	Ala	residue_name	Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis.	RESULTS
+138	147	autolysis	ptm	Whereas Asn can to some degree replace Asp166 due to its carbonyl group in the side chain, Ala at this position was found to prevent both autolysis and catalysis.	RESULTS
+27	33	Asp166	residue_name_number	These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis.	RESULTS
+38	44	Ser129	residue_name_number	These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis.	RESULTS
+110	114	Thr1	residue_name_number	These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis.	RESULTS
+123	132	autolysis	ptm	These results suggest that Asp166 and Ser129 function as a proton shuttle and affect the protonation state of Thr1N during autolysis and catalysis.	RESULTS
+0	12	Substitution	experimental_method	Substitution of the active-site Thr1 by Cys	RESULTS
+20	31	active-site	site	Substitution of the active-site Thr1 by Cys	RESULTS
+32	36	Thr1	residue_name_number	Substitution of the active-site Thr1 by Cys	RESULTS
+40	43	Cys	residue_name	Substitution of the active-site Thr1 by Cys	RESULTS
+0	8	Mutation	experimental_method	Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.	RESULTS
+12	16	Thr1	residue_name_number	Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.	RESULTS
+20	23	Cys	residue_name	Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.	RESULTS
+40	54	20S proteasome	complex_assembly	Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.	RESULTS
+64	72	archaeon	taxonomy_domain	Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.	RESULTS
+73	87	T. acidophilum	species	Mutation of Thr1 to Cys inactivates the 20S proteasome from the archaeon T. acidophilum.	RESULTS
+3	8	yeast	taxonomy_domain	In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure.	RESULTS
+15	23	mutation	experimental_method	In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure.	RESULTS
+90	100	propeptide	structure_element	In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure.	RESULTS
+137	152	X-ray structure	evidence	In yeast, this mutation causes a strong growth defect (Fig. 4a and Table 1), although the propeptide is hydrolysed, as shown here by its X-ray structure.	RESULTS
+18	20	β5	protein	In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c).	RESULTS
+53	60	cleaved	protein_state	In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c).	RESULTS
+61	71	propeptide	structure_element	In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c).	RESULTS
+72	83	still bound	protein_state	In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c).	RESULTS
+91	116	substrate-binding channel	site	In one of the two β5 subunits, however, we found the cleaved propeptide still bound in the substrate-binding channel (Fig. 4c).	RESULTS
+0	7	His(-2)	residue_name_number	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+21	30	S2 pocket	site	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+53	64	β5-T1A-K81R	mutant	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+65	71	mutant	protein_state	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+108	118	propeptide	structure_element	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+126	129	T1C	mutant	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+130	136	mutant	protein_state	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+147	167	antiparallel β-sheet	structure_element	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+211	216	MG132	chemical	His(-2) occupies the S2 pocket like observed for the β5-T1A-K81R mutant, but in contrast to the latter, the propeptide in the T1C mutant adopts an antiparallel β-sheet conformation as known from inhibitors like MG132 (Fig. 4c–e and Supplementary Fig. 9b).	RESULTS
+37	40	T1C	mutant	On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.	RESULTS
+41	47	mutant	protein_state	On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.	RESULTS
+56	66	propeptide	structure_element	On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.	RESULTS
+93	104	active site	site	On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.	RESULTS
+122	131	autolysis	ptm	On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.	RESULTS
+183	198	crystallization	experimental_method	On the basis of the phenotype of the T1C mutant and the propeptide remnant identified in its active site, we suppose that autolysis is retarded and may not have been completed before crystallization.	RESULTS
+42	44	β5	protein	Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits.	RESULTS
+65	67	CP	complex_assembly	Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits.	RESULTS
+107	117	propeptide	structure_element	Owing to the unequal positions of the two β5 subunits within the CP in the crystal lattice, maturation and propeptide displacement may occur at different timescales in the two subunits.	RESULTS
+8	29	propeptide hydrolysis	ptm	Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a).	RESULTS
+35	41	β5-T1C	mutant	Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a).	RESULTS
+42	53	active site	site	Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a).	RESULTS
+57	79	catalytically inactive	protein_state	Despite propeptide hydrolysis, the β5-T1C active site is catalytically inactive (Fig. 4b and Supplementary Fig. 9a).	RESULTS
+14	30	soaking crystals	experimental_method	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+40	42	CP	complex_assembly	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+54	64	bortezomib	chemical	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+68	79	carfilzomib	chemical	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+98	100	β1	protein	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+105	107	β2	protein	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+108	120	active sites	site	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+140	146	β5-T1C	mutant	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+147	166	proteolytic centres	site	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+167	177	unmodified	protein_state	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+230	237	cleaved	protein_state	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+238	248	propeptide	structure_element	In agreement, soaking crystals with the CP inhibitors bortezomib or carfilzomib modifies only the β1 and β2 active sites, while leaving the β5-T1C proteolytic centres unmodified even though they are only partially occupied by the cleaved propeptide remnant.	RESULTS
+14	29	structural data	evidence	Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b).	RESULTS
+61	65	Cys1	residue_name_number	Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b).	RESULTS
+127	131	Thr1	residue_name_number	Moreover, the structural data reveal that the thiol group of Cys1 is rotated by 74° with respect to the hydroxyl side chain of Thr1 (Fig. 4f and Supplementary Fig. 9b).	RESULTS
+73	78	Lys33	residue_name_number	Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1.	RESULTS
+82	84	WT	protein_state	Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1.	RESULTS
+85	88	CPs	complex_assembly	Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1.	RESULTS
+104	108	Cys1	residue_name_number	Consequently, the hydrogen bond bridging the active-site nucleophile and Lys33 in WT CPs is broken with Cys1.	RESULTS
+13	40	2FO–FC electron-density map	evidence	Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered.	RESULTS
+48	51	T1C	mutant	Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered.	RESULTS
+52	58	mutant	protein_state	Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered.	RESULTS
+79	84	Lys33	residue_name_number	Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered.	RESULTS
+91	101	disordered	protein_state	Notably, the 2FO–FC electron-density map of the T1C mutant also indicates that Lys33NH2 is disordered.	RESULTS
+90	95	Lys33	residue_name_number	Together, these observations suggest that efficient peptide-bond hydrolysis requires that Lys33NH2 hydrogen bonds to the active site nucleophile.	RESULTS
+15	18	Thr	residue_name	The benefit of Thr over Ser as the active-site nucleophile	RESULTS
+24	27	Ser	residue_name	The benefit of Thr over Ser as the active-site nucleophile	RESULTS
+4	15	proteasomes	complex_assembly	All proteasomes strictly employ threonine as the active-site residue instead of serine.	RESULTS
+16	31	strictly employ	protein_state	All proteasomes strictly employ threonine as the active-site residue instead of serine.	RESULTS
+32	41	threonine	residue_name	All proteasomes strictly employ threonine as the active-site residue instead of serine.	RESULTS
+49	68	active-site residue	site	All proteasomes strictly employ threonine as the active-site residue instead of serine.	RESULTS
+80	86	serine	residue_name	All proteasomes strictly employ threonine as the active-site residue instead of serine.	RESULTS
+62	68	β5-T1S	mutant	To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1).	RESULTS
+69	75	mutant	protein_state	To investigate the reason for this singularity, we analysed a β5-T1S mutant, which is viable but suffers from growth defects (Fig. 4a and Table 1).	RESULTS
+0	15	Activity assays	experimental_method	Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).	RESULTS
+25	27	β5	protein	Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).	RESULTS
+47	59	Suc-LLVY-AMC	chemical	Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).	RESULTS
+104	107	T1S	mutant	Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).	RESULTS
+108	114	mutant	protein_state	Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).	RESULTS
+150	152	WT	protein_state	Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).	RESULTS
+153	164	proteasomes	complex_assembly	Activity assays with the β5-specific substrate Suc-LLVY-AMC demonstrated that the ChT-L activity of the T1S mutant is reduced by 40–45% compared with WT proteasomes depending on the incubation temperature (Fig. 4b and Supplementary Fig. 9c).	RESULTS
+39	48	Z-GGL-pNA	chemical	By contrast, turnover of the substrate Z-GGL-pNA, used to monitor ChT-L activity in situ but in a less quantitative fashion, is not detectably impaired (Supplementary Fig. 9a).	RESULTS
+0	17	Crystal structure	evidence	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+34	40	β5-T1S	mutant	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+41	47	mutant	protein_state	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+58	78	precursor processing	ptm	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+94	108	ligand-complex	complex_assembly	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+109	119	structures	evidence	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+125	135	bortezomib	chemical	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+140	151	carfilzomib	chemical	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+197	201	Ser1	residue_name_number	Crystal structure analysis of the β5-T1S mutant confirmed precursor processing (Fig. 4g), and ligand-complex structures with bortezomib and carfilzomib unambiguously corroborated the reactivity of Ser1 (Fig. 5).	RESULTS
+13	16	apo	protein_state	However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g).	RESULTS
+17	34	crystal structure	evidence	However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g).	RESULTS
+49	53	Ser1	residue_name_number	However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g).	RESULTS
+80	105	substrate-binding channel	site	However, the apo crystal structure revealed that Ser1Oγ is turned away from the substrate-binding channel (Fig. 4g).	RESULTS
+14	18	Thr1	residue_name_number	Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°.	RESULTS
+24	26	WT	protein_state	Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°.	RESULTS
+27	29	CP	complex_assembly	Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°.	RESULTS
+30	40	structures	evidence	Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°.	RESULTS
+42	46	Ser1	residue_name_number	Compared with Thr1Oγ in WT CP structures, Ser1Oγ is rotated by 60°.	RESULTS
+30	34	Ser1	residue_name_number	Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1.	RESULTS
+60	65	Lys33	residue_name_number	Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1.	RESULTS
+179	183	Thr1	residue_name_number	Because both conformations of Ser1Oγ are hydrogen-bonded to Lys33NH2 (Fig. 4h), the relay system is capable of hydrolysing peptide substrates, albeit at lower rates compared with Thr1.	RESULTS
+4	23	active-site residue	site	The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h).	RESULTS
+24	28	Thr1	residue_name_number	The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h).	RESULTS
+119	123	Thr3	residue_name_number	The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h).	RESULTS
+128	133	Ala46	residue_name_number	The active-site residue Thr1 is fixed in its position, as its methyl group is engaged in hydrophobic interactions with Thr3 and Ala46 (Fig. 4h).	RESULTS
+36	40	Thr1	residue_name_number	Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1.	RESULTS
+139	143	Ser1	residue_name_number	Consequently, the hydroxyl group of Thr1 requires no reorientation before substrate cleavage and is thus more catalytically efficient than Ser1.	RESULTS
+62	65	T1S	mutant	In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a).	RESULTS
+66	72	mutant	protein_state	In agreement, at an elevated growing temperature of 37 °C the T1S mutant is unable to grow (Fig. 4a).	RESULTS
+14	20	mutant	protein_state	In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5).	RESULTS
+21	31	proteasome	complex_assembly	In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5).	RESULTS
+55	65	proteasome	complex_assembly	In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5).	RESULTS
+80	90	bortezomib	chemical	In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5).	RESULTS
+106	117	carfilzomib	chemical	In vitro, the mutant proteasome is less susceptible to proteasome inhibition by bortezomib (3.7-fold) and carfilzomib (1.8-fold; Fig. 5).	RESULTS
+14	31	inhibitor complex	complex_assembly	Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.	RESULTS
+32	42	structures	evidence	Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.	RESULTS
+94	96	WT	protein_state	Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.	RESULTS
+97	100	yCP	complex_assembly	Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.	RESULTS
+101	111	structures	evidence	Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.	RESULTS
+113	137	with the same inhibitors	protein_state	Nevertheless, inhibitor complex structures indicate identical binding modes compared with the WT yCP structures, with the same inhibitors.	RESULTS
+13	21	affinity	evidence	Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein.	RESULTS
+42	53	carfilzomib	chemical	Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein.	RESULTS
+106	131	substrate-binding channel	site	Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein.	RESULTS
+151	161	bortezomib	chemical	Notably, the affinity of the tetrapeptide carfilzomib is less impaired, as it is better stabilized in the substrate-binding channel than the dipeptide bortezomib, which lacks a defined P3 site and has only a few interactions with the surrounding protein.	RESULTS
+11	30	mean residence time	evidence	Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased.	RESULTS
+34	45	carfilzomib	chemical	Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased.	RESULTS
+53	64	active site	site	Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased.	RESULTS
+123	127	Ser1	residue_name_number	Hence, the mean residence time of carfilzomib at the active site is prolonged and the probability to covalently react with Ser1 is increased.	RESULTS
+89	98	threonine	residue_name	Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.).	RESULTS
+133	144	proteasomes	complex_assembly	Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.).	RESULTS
+189	214	proteasome-like proteases	protein_type	Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.).	RESULTS
+223	227	HslV	protein	Considered together, these results provide a plausible explanation for the invariance of threonine as the active-site nucleophile in proteasomes in all three domains of life, as well as in proteasome-like proteases such as HslV (ref.).	RESULTS
+4	18	20S proteasome	complex_assembly	The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized.	DISCUSS
+19	21	CP	complex_assembly	The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized.	DISCUSS
+35	57	non-lysosomal protease	protein_type	The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized.	DISCUSS
+61	71	eukaryotic	taxonomy_domain	The 20S proteasome CP is the major non-lysosomal protease in eukaryotic cells, and its assembly is highly organized.	DISCUSS
+4	13	β-subunit	protein	The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex.	DISCUSS
+14	25	propeptides	structure_element	The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex.	DISCUSS
+48	50	β5	protein	The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex.	DISCUSS
+107	109	CP	complex_assembly	The β-subunit propeptides, particularly that of β5, are key factors that help drive proper assembly of the CP complex.	DISCUSS
+59	63	Thr1	residue_name_number	In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation.	DISCUSS
+78	91	N-acetylation	ptm	In addition, they prevent irreversible inactivation of the Thr1 N terminus by N-acetylation.	DISCUSS
+17	28	prosegments	structure_element	By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.	DISCUSS
+32	42	β subunits	protein	By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.	DISCUSS
+63	71	archaeal	taxonomy_domain	By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.	DISCUSS
+72	82	proteasome	complex_assembly	By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.	DISCUSS
+107	131	heterologously expressed	experimental_method	By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.	DISCUSS
+135	151	Escherichia coli	species	By contrast, the prosegments of β subunits are dispensable for archaeal proteasome assembly, at least when heterologously expressed in Escherichia coli.	DISCUSS
+3	13	eukaryotes	taxonomy_domain	In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated.	DISCUSS
+52	54	β1	protein	In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated.	DISCUSS
+59	61	β2	protein	In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated.	DISCUSS
+62	73	propeptides	structure_element	In eukaryotes, deletion of or failure to cleave the β1 and β2 propeptides is well tolerated.	DISCUSS
+9	19	removal of	experimental_method	However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects.	DISCUSS
+24	26	β5	protein	However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects.	DISCUSS
+27	37	prosegment	structure_element	However, removal of the β5 prosegment or any interference with its cleavage causes severe phenotypic defects.	DISCUSS
+71	73	β5	protein	These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP.	DISCUSS
+74	84	propeptide	structure_element	These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP.	DISCUSS
+100	102	β5	protein	These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP.	DISCUSS
+103	114	active site	site	These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP.	DISCUSS
+150	160	eukaryotic	taxonomy_domain	These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP.	DISCUSS
+161	163	CP	complex_assembly	These observations highlight the unique function and importance of the β5 propeptide as well as the β5 active site for maturation and function of the eukaryotic CP.	DISCUSS
+27	44	atomic structures	evidence	Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide.	DISCUSS
+56	62	β5-T1A	mutant	Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide.	DISCUSS
+135	137	β5	protein	Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide.	DISCUSS
+138	148	propeptide	structure_element	Here we have described the atomic structures of various β5-T1A mutants, which allowed for the first time visualization of the residual β5 propeptide.	DISCUSS
+17	21	(-2)	residue_number	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+50	60	propeptide	structure_element	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+80	87	Gly(-1)	residue_name_number	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+98	108	structures	evidence	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+158	162	Thr1	residue_name_number	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+197	207	tight turn	structure_element	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+225	235	prosegment	structure_element	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+247	249	β1	protein	Depending on the (-2) residue we observed various propeptide conformations, but Gly(-1) is in all structures perfectly located for the nucleophilic attack by Thr1Oγ, although it does not adopt the tight turn observed for the prosegment of subunit β1.	DISCUSS
+57	64	Gly(-1)	residue_name_number	From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis.	DISCUSS
+69	73	Thr1	residue_name_number	From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis.	DISCUSS
+118	129	active site	site	From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis.	DISCUSS
+147	156	autolysis	ptm	From these data we conclude that only the positioning of Gly(-1) and Thr1 as well as the integrity of the proteasomal active site are required for autolysis.	DISCUSS
+30	43	N-acetylation	ptm	In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group.	DISCUSS
+51	55	Thr1	residue_name_number	In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group.	DISCUSS
+88	92	Thr1	residue_name_number	In this regard, inappropriate N-acetylation of the Thr1 N terminus cannot be removed by Thr1Oγ due to the rotational freedom and flexibility of the acetyl group.	DISCUSS
+4	14	propeptide	structure_element	The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2).	DISCUSS
+43	68	substrate-binding channel	site	The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2).	DISCUSS
+90	97	Gly(-1)	residue_name_number	The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2).	DISCUSS
+162	166	(-2)	residue_number	The propeptide needs some anchoring in the substrate-binding channel to properly position Gly(-1), but this seems to be independent of the orientation of residue (-2).	DISCUSS
+28	30	CP	complex_assembly	Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic.	DISCUSS
+112	131	propeptide cleavage	ptm	Autolytic activation of the CP constitutes one of the final steps of proteasome biogenesis, but the trigger for propeptide cleavage had remained enigmatic.	DISCUSS
+29	38	CP:ligand	complex_assembly	On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture.	DISCUSS
+146	156	proteasome	complex_assembly	On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture.	DISCUSS
+157	181	active-site architecture	site	On the basis of the numerous CP:ligand complexes solved during the past 18 years and in the current study, we provide a revised interpretation of proteasome active-site architecture.	DISCUSS
+13	28	catalytic triad	site	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+37	48	active site	site	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+56	58	CP	complex_assembly	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+82	86	Thr1	residue_name_number	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+88	93	Lys33	residue_name_number	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+98	101	Asp	residue_name	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+102	107	Glu17	residue_name_number	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+162	172	eukaryotic	taxonomy_domain	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+174	183	bacterial	taxonomy_domain	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+188	196	archaeal	taxonomy_domain	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+197	207	proteasome	complex_assembly	We propose a catalytic triad for the active site of the CP consisting of residues Thr1, Lys33 and Asp/Glu17, which are conserved among all proteolytically active eukaryotic, bacterial and archaeal proteasome subunits.	DISCUSS
+0	5	Lys33	residue_name_number	Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å).	DISCUSS
+58	79	autocatalytic removal	ptm	Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å).	DISCUSS
+87	98	propeptides	structure_element	Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å).	DISCUSS
+147	152	Asp17	residue_name_number	Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å).	DISCUSS
+163	168	Lys33	residue_name_number	Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å).	DISCUSS
+255	260	Lys33	residue_name_number	Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å).	DISCUSS
+265	270	Asp17	residue_name_number	Lys33NH2 is expected to act as the proton acceptor during autocatalytic removal of the propeptides, as well as during substrate proteolysis, while Asp17Oδ orients Lys33NH2 and makes it more prone to protonation by raising its pKa (hydrogen bond distance: Lys33NH3+–Asp17Oδ: 2.9 Å).	DISCUSS
+19	29	proteasome	complex_assembly	Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase.	DISCUSS
+33	50	Thr–Lys–Asp triad	site	Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase.	DISCUSS
+68	82	L-asparaginase	protein_type	Analogously to the proteasome, a Thr–Lys–Asp triad is also found in L-asparaginase.	DISCUSS
+107	110	Lys	residue_name	Thus, specific protein surroundings can significantly alter the chemical properties of amino acids such as Lys to function as an acid–base catalyst.	DISCUSS
+36	47	active site	site	In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d).	DISCUSS
+72	76	Thr1	residue_name_number	In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d).	DISCUSS
+207	231	endoproteolytic cleavage	ptm	In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d).	DISCUSS
+235	239	Thr1	residue_name_number	In this new view of the proteasomal active site, the positively charged Thr1NH3+-terminus hydrogen bonds to the amide nitrogen of incoming peptide substrates and stabilizes as well as activates them for the endoproteolytic cleavage by Thr1Oγ (Fig. 3d).	DISCUSS
+51	55	Thr1	residue_name_number	Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.).	DISCUSS
+127	140	fellutamide B	chemical	Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.).	DISCUSS
+149	161	α-ketoamides	chemical	Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.).	DISCUSS
+163	179	homobelactosin C	chemical	Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.).	DISCUSS
+191	208	salinosporamide A	chemical	Consistent with this model, the positively charged Thr1 N terminus is engaged in hydrogen bonds with inhibitory compounds like fellutamide B (ref.), α-ketoamides, homobelactosin C (ref.) and salinosporamide A (ref.).	DISCUSS
+47	56	omuralide	chemical	Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+.	DISCUSS
+60	64	Thr1	residue_name_number	Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+.	DISCUSS
+123	127	Thr1	residue_name_number	Furthermore, opening of the β-lactone compound omuralide by Thr1 creates a C3-hydroxyl group, whose proton originates from Thr1NH3+.	DISCUSS
+24	28	Thr1	residue_name_number	The resulting uncharged Thr1NH2 is hydrogen-bridged to the C3-OH group.	DISCUSS
+14	25	acetylation	ptm	In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons.	DISCUSS
+33	37	Thr1	residue_name_number	In agreement, acetylation of the Thr1 N terminus irreversibly blocks hydrolytic activity, and binding of substrates is prevented for steric reasons.	DISCUSS
+50	54	Thr1	residue_name_number	By acting as a proton donor during catalysis, the Thr1 N terminus may also favour cleavage of substrate peptide bonds (Fig. 3d).	DISCUSS
+98	108	proteasome	complex_assembly	Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function.	DISCUSS
+114	118	Thr1	residue_name_number	Cleavage of the scissile peptide bond requires protonation of the emerging free amine, and in the proteasome, the Thr1 amine group is likely to assume this function.	DISCUSS
+13	17	Thr1	residue_name_number	Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2.	DISCUSS
+226	230	Thr1	residue_name_number	Analogously, Thr1NH3+ might promote the bivalent reaction mode of epoxyketone inhibitors by protonating the epoxide moiety to create a positively charged trivalent oxygen atom that is subsequently nucleophilically attacked by Thr1NH2.	DISCUSS
+7	16	autolysis	ptm	During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived.	DISCUSS
+21	25	Thr1	residue_name_number	During autolysis the Thr1 N terminus is engaged in a hydroxyoxazolidine ring intermediate (Fig. 3d), which is unstable and short-lived.	DISCUSS
+60	64	Thr1	residue_name_number	Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+.	DISCUSS
+98	115	aspartic acid 166	residue_name_number	Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+.	DISCUSS
+120	126	Ser129	residue_name_number	Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+.	DISCUSS
+138	142	Thr1	residue_name_number	Breakdown of this tetrahedral transition state releases the Thr1 N terminus that is protonated by aspartic acid 166 via Ser129OH to yield Thr1NH3+.	DISCUSS
+13	19	Ser129	residue_name_number	The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state.	DISCUSS
+24	30	Asp166	residue_name_number	The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state.	DISCUSS
+73	77	Thr1	residue_name_number	The residues Ser129 and Asp166 are expected to increase the pKa value of Thr1N, thereby favouring its charged state.	DISCUSS
+67	75	mutation	experimental_method	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+76	81	D166A	mutant	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+91	100	autolysis	ptm	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+108	116	archaeal	taxonomy_domain	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+117	119	CP	complex_assembly	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+128	136	exchange	experimental_method	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+137	142	D166N	mutant	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+177	182	yeast	taxonomy_domain	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+183	185	CP	complex_assembly	Consistent with playing an essential role in proton shuttling, the mutation D166A prevents autolysis of the archaeal CP and the exchange D166N impairs catalytic activity of the yeast CP about 60%.	DISCUSS
+4	12	mutation	experimental_method	The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2).	DISCUSS
+13	18	D166N	mutant	The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2).	DISCUSS
+37	41	Thr1	residue_name_number	The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2).	DISCUSS
+116	120	Thr1	residue_name_number	The mutation D166N lowers the pKa of Thr1N, which is thus more likely to exist in the uncharged deprotonated state (Thr1NH2).	DISCUSS
+79	87	β5-D166N	mutant	This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other.	DISCUSS
+88	94	mutant	protein_state	This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other.	DISCUSS
+150	161	carfilzomib	chemical	This interpretation agrees with the strongly reduced catalytic activity of the β5-D166N mutant on the one hand, and the ability to react readily with carfilzomib on the other.	DISCUSS
+11	21	proteasome	complex_assembly	Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis.	DISCUSS
+48	60	second triad	site	Hence, the proteasome can be viewed as having a second triad that is essential for efficient proteolysis.	DISCUSS
+6	11	Lys33	residue_name_number	While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.	DISCUSS
+19	24	Asp17	residue_name_number	While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.	DISCUSS
+59	63	Thr1	residue_name_number	While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.	DISCUSS
+85	91	Ser129	residue_name_number	While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.	DISCUSS
+98	104	Asp166	residue_name_number	While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.	DISCUSS
+156	160	Thr1	residue_name_number	While Lys33NH2 and Asp17Oδ are required to deprotonate the Thr1 hydroxyl side chain, Ser129OH and Asp166OH serve to protonate the N-terminal amine group of Thr1.	DISCUSS
+28	32	Thr1	residue_name_number	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+33	38	Lys33	residue_name_number	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+39	44	Asp17	residue_name_number	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+45	60	catalytic triad	site	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+62	83	crystallographic data	evidence	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+91	115	proteolytically inactive	protein_state	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+116	122	β5-T1C	mutant	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+123	129	mutant	protein_state	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+166	171	Lys33	residue_name_number	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+179	183	Cys1	residue_name_number	In accord with the proposed Thr1–Lys33–Asp17 catalytic triad, crystallographic data on the proteolytically inactive β5-T1C mutant demonstrate that the interaction of Lys33NH2 and Cys1 is broken.	DISCUSS
+18	21	Cys	residue_name	However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time.	DISCUSS
+54	64	propeptide	structure_element	However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time.	DISCUSS
+78	85	cleaved	protein_state	However, owing to Cys being a strong nucleophile, the propeptide can still be cleaved off over time.	DISCUSS
+48	57	autolysis	ptm	While only one single turnover is necessary for autolysis, continuous enzymatic activity is required for significant and detectable substrate hydrolysis.	DISCUSS
+16	29	Ntn hydrolase	protein_type	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+30	48	penicillin acylase	protein_type	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+50	62	substitution	experimental_method	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+70	79	catalytic	protein_state	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+91	94	Ser	residue_name	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+106	109	Cys	residue_name	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+115	126	inactivates	protein_state	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+131	137	enzyme	protein_type	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+156	176	precursor processing	ptm	Notably, in the Ntn hydrolase penicillin acylase, substitution of the catalytic N-terminal Ser residue by Cys also inactivates the enzyme but still enables precursor processing.	DISCUSS
+23	25	CP	complex_assembly	To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally.	DISCUSS
+47	56	threonine	residue_name	To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally.	DISCUSS
+64	83	active-site residue	site	To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally.	DISCUSS
+95	101	β5-T1S	mutant	To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally.	DISCUSS
+102	108	mutant	protein_state	To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally.	DISCUSS
+116	119	yCP	complex_assembly	To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally.	DISCUSS
+141	171	biochemically and structurally	experimental_method	To investigate why the CP specifically employs threonine as its active-site residue, we used a β5-T1S mutant of the yCP and characterized it biochemically and structurally.	DISCUSS
+0	15	Activity assays	experimental_method	Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC.	DISCUSS
+25	31	β5-T1S	mutant	Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC.	DISCUSS
+32	38	mutant	protein_state	Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC.	DISCUSS
+68	80	Suc-LLVY-AMC	chemical	Activity assays with the β5-T1S mutant revealed reduced turnover of Suc-LLVY-AMC.	DISCUSS
+48	54	β5-T1S	mutant	We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.	DISCUSS
+55	61	mutant	protein_state	We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.	DISCUSS
+62	65	yCP	complex_assembly	We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.	DISCUSS
+112	122	proteasome	complex_assembly	We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.	DISCUSS
+134	144	bortezomib	chemical	We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.	DISCUSS
+149	160	carfilzomib	chemical	We also observed slightly lower affinity of the β5-T1S mutant yCP for the Food and Drug Administration-approved proteasome inhibitors bortezomib and carfilzomib.	DISCUSS
+0	19	Structural analyses	evidence	Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.	DISCUSS
+52	55	T1S	mutant	Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.	DISCUSS
+56	62	mutant	protein_state	Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.	DISCUSS
+98	111	strict use of	protein_state	Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.	DISCUSS
+112	115	Thr	residue_name	Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.	DISCUSS
+128	139	proteasomes	complex_assembly	Structural analyses support these findings with the T1S mutant and provide an explanation for the strict use of Thr residues in proteasomes.	DISCUSS
+0	4	Thr1	residue_name_number	Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ).	DISCUSS
+29	40	active site	site	Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ).	DISCUSS
+97	102	Ala46	residue_name_number	Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ).	DISCUSS
+109	114	Lys33	residue_name_number	Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ).	DISCUSS
+139	143	Thr3	residue_name_number	Thr1 is well anchored in the active site by hydrophobic interactions of its Cγ methyl group with Ala46 (Cβ), Lys33 (carbon side chain) and Thr3 (Cγ).	DISCUSS
+9	31	proteolytically active	protein_state	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+32	42	proteasome	complex_assembly	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+57	64	archaea	taxonomy_domain	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+66	71	yeast	taxonomy_domain	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+76	83	mammals	taxonomy_domain	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+161	164	Thr	residue_name	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+168	171	Ile	residue_name	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+184	185	3	residue_number	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+267	271	Thr1	residue_name_number	Notably, proteolytically active proteasome subunits from archaea, yeast and mammals, including constitutive, immuno- and thymoproteasome subunits, either encode Thr or Ile at position 3, indicating the importance of the Cγ for fixing the position of the nucleophilic Thr1.	DISCUSS
+15	19	Thr1	residue_name_number	In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h).	DISCUSS
+43	47	Ser1	residue_name_number	In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h).	DISCUSS
+77	81	Thr1	residue_name_number	In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h).	DISCUSS
+107	109	WT	protein_state	In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h).	DISCUSS
+110	116	enzyme	complex_assembly	In contrast to Thr1, the hydroxyl group of Ser1 occupies the position of the Thr1 methyl side chain in the WT enzyme, which requires its reorientation relative to the substrate to allow cleavage (Fig. 4g,h).	DISCUSS
+16	35	threonine aspartase	protein_type	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+36	44	Taspase1	protein	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+46	54	mutation	experimental_method	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+62	73	active-site	site	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+74	80	Thr234	residue_name_number	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+84	87	Ser	residue_name	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+154	160	Thr234	residue_name_number	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+168	170	WT	protein_state	Notably, in the threonine aspartase Taspase1, mutation of the active-site Thr234 to Ser also places the side chain in the position of the methyl group of Thr234 in the WT, thereby reducing catalytic activity.	DISCUSS
+24	30	serine	residue_name	Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.	DISCUSS
+31	37	mutant	protein_state	Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.	DISCUSS
+41	47	active	protein_state	Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.	DISCUSS
+49	58	threonine	residue_name	Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.	DISCUSS
+99	109	proteasome	complex_assembly	Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.	DISCUSS
+110	121	active site	site	Similarly, although the serine mutant is active, threonine is more efficient in the context of the proteasome active site.	DISCUSS
+27	36	threonine	residue_name	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+45	55	proteasome	complex_assembly	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+56	67	active site	site	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+172	184	conservation	protein_state	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+192	196	Thr1	residue_name_number	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+212	223	proteasomes	complex_assembly	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+229	237	bacteria	taxonomy_domain	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+241	251	eukaryotes	taxonomy_domain	The greater suitability of threonine for the proteasome active site, which has been noted in biochemical as well as in kinetic studies, constitutes a likely reason for the conservation of the Thr1 residue in all proteasomes from bacteria to eukaryotes.	DISCUSS
+28	39	propeptides	structure_element	Conformation of proteasomal propeptides.	FIG
+4	28	Structural superposition	experimental_method	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+36	42	β1-T1A	mutant	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+43	53	propeptide	structure_element	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+62	69	matured	protein_state	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+70	72	WT	protein_state	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+73	75	β1	protein	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+76	87	active-site	site	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+88	92	Thr1	residue_name_number	(a) Structural superposition of the β1-T1A propeptide and the matured WT β1 active-site Thr1.	FIG
+18	30	(-5) to (-1)	residue_range	Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed.	FIG
+38	44	β1-T1A	mutant	Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed.	FIG
+45	55	propeptide	structure_element	Only the residues (-5) to (-1) of the β1-T1A propeptide are displayed.	FIG
+29	50	S1 specificity pocket	site	The major determinant of the S1 specificity pocket, residue 45, is depicted.	FIG
+60	62	45	residue_number	The major determinant of the S1 specificity pocket, residue 45, is depicted.	FIG
+29	50	S1 specificity pocket	site	The major determinant of the S1 specificity pocket, residue 45, is depicted.	FIG
+60	62	45	residue_number	The major determinant of the S1 specificity pocket, residue 45, is depicted.	FIG
+31	38	Gly(-1)	residue_name_number	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+43	47	Ala1	residue_name_number	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+55	65	propeptide	structure_element	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+75	80	G(-1)	residue_name_number	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+133	142	processed	protein_state	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+143	145	WT	protein_state	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+146	157	active-site	site	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+158	162	Thr1	residue_name_number	Note the tight conformation of Gly(-1) and Ala1 before propeptide removal (G(-1) turn; cyan double arrow) compared with the relaxed, processed WT active-site Thr1 (red double arrow).	FIG
+40	44	Thr1	residue_name_number	The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1).	FIG
+80	87	Gly(-1)	residue_name_number	The black arrow indicates the attack of Thr1Oγ onto the carbonyl carbon atom of Gly(-1).	FIG
+4	28	Structural superposition	experimental_method	(b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.	FIG
+36	42	β1-T1A	mutant	(b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.	FIG
+43	53	propeptide	structure_element	(b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.	FIG
+62	68	β2-T1A	mutant	(b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.	FIG
+69	79	propeptide	structure_element	(b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.	FIG
+155	159	Ala1	residue_name_number	(b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.	FIG
+164	171	Gly(-1)	residue_name_number	(b) Structural superposition of the β1-T1A propeptide and the β2-T1A propeptide highlights subtle differences in their conformations, but illustrates that Ala1 and Gly(-1) match well.	FIG
+0	7	Thr(-2)	residue_name_number	Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line).	FIG
+32	39	Gly(-1)	residue_name_number	Thr(-2)OH is hydrogen-bonded to Gly(-1)O (∼2.8 Å; black dashed line).	FIG
+4	28	Structural superposition	experimental_method	(c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation.	FIG
+36	42	β1-T1A	mutant	(c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation.	FIG
+48	54	β2-T1A	mutant	(c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation.	FIG
+63	74	β5-T1A-K81R	mutant	(c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation.	FIG
+75	85	propeptide	structure_element	(c) Structural superposition of the β1-T1A, the β2-T1A and the β5-T1A-K81R propeptide remnants depict their differences in conformation.	FIG
+14	18	(-2)	residue_number	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+26	28	β1	protein	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+33	35	β2	protein	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+36	47	prosegments	structure_element	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+56	65	S1 pocket	site	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+67	74	His(-2)	residue_name_number	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+82	84	β5	protein	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+85	95	propeptide	structure_element	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+109	118	S2 pocket	site	While residue (-2) of the β1 and β2 prosegments fit the S1 pocket, His(-2) of the β5 propeptide occupies the S2 pocket.	FIG
+56	63	Gly(-1)	residue_name_number	Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ.	FIG
+113	117	Thr1	residue_name_number	Nonetheless, in all mutants the carbonyl carbon atom of Gly(-1) is ideally placed for the nucleophilic attack by Thr1Oγ.	FIG
+26	33	Thr(-2)	residue_name_number	The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line.	FIG
+40	47	Gly(-1)	residue_name_number	The hydrogen bond between Thr(-2)OH and Gly(-1)O (∼2.8 Å) is indicated by a black dashed line.	FIG
+0	9	Mutations	experimental_method	Mutations of residue (-2) and their influence on propeptide conformation and autolysis.	FIG
+21	25	(-2)	residue_number	Mutations of residue (-2) and their influence on propeptide conformation and autolysis.	FIG
+49	59	propeptide	structure_element	Mutations of residue (-2) and their influence on propeptide conformation and autolysis.	FIG
+77	86	autolysis	ptm	Mutations of residue (-2) and their influence on propeptide conformation and autolysis.	FIG
+4	28	Structural superposition	experimental_method	(a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide.	FIG
+36	42	β1-T1A	mutant	(a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide.	FIG
+43	53	propeptide	structure_element	(a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide.	FIG
+62	75	β5-H(-2)L-T1A	mutant	(a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide.	FIG
+76	82	mutant	protein_state	(a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide.	FIG
+83	93	propeptide	structure_element	(a) Structural superposition of the β1-T1A propeptide and the β5-H(-2)L-T1A mutant propeptide.	FIG
+4	8	(-2)	residue_number	The (-2) residues of both prosegments point into the S1 pocket.	FIG
+26	37	prosegments	structure_element	The (-2) residues of both prosegments point into the S1 pocket.	FIG
+53	62	S1 pocket	site	The (-2) residues of both prosegments point into the S1 pocket.	FIG
+4	28	Structural superposition	experimental_method	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+36	38	β5	protein	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+39	50	propeptides	structure_element	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+58	71	β5-H(-2)L-T1A	mutant	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+73	86	β5-H(-2)T-T1A	mutant	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+88	106	β5-(H-2)A-T1A-K81R	mutant	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+111	122	β5-T1A-K81R	mutant	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+123	129	mutant	protein_state	(b) Structural superposition of the β5 propeptides in the β5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+130	141	proteasomes	complex_assembly	(b) Structural superposition of the β5 propeptides in the ��5-H(-2)L-T1A, β5-H(-2)T-T1A, β5-(H-2)A-T1A-K81R and β5-T1A-K81R mutant proteasomes.	FIG
+19	31	(-2) to (-4)	residue_range	While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions.	FIG
+60	67	Gly(-1)	residue_name_number	While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions.	FIG
+72	76	Ala1	residue_name_number	While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions.	FIG
+96	106	structures	evidence	While the residues (-2) to (-4) vary in their conformation, Gly(-1) and Ala1 are located in all structures at the same positions.	FIG
+4	28	Structural superposition	experimental_method	(c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide.	FIG
+36	42	β2-T1A	mutant	(c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide.	FIG
+43	53	propeptide	structure_element	(c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide.	FIG
+62	75	β5-H(-2)T-T1A	mutant	(c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide.	FIG
+76	82	mutant	protein_state	(c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide.	FIG
+83	93	propeptide	structure_element	(c) Structural superposition of the β2-T1A propeptide and the β5-H(-2)T-T1A mutant propeptide.	FIG
+4	8	(-2)	residue_number	The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line).	FIG
+26	37	prosegments	structure_element	The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line).	FIG
+53	62	S1 pocket	site	The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line).	FIG
+73	80	Thr(-2)	residue_name_number	The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line).	FIG
+86	88	β2	protein	The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line).	FIG
+116	123	Gly(-1)	residue_name_number	The (-2) residues of both prosegments point into the S1 pocket, but only Thr(-2)OH of β2 forms a hydrogen bridge to Gly(-1)O (black dashed line).	FIG
+4	28	Structural superposition	experimental_method	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+36	43	matured	protein_state	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+44	46	β2	protein	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+47	58	active site	site	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+64	66	WT	protein_state	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+67	73	β2-T1A	mutant	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+74	84	propeptide	structure_element	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+93	102	β2-T(-2)V	mutant	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+103	109	mutant	protein_state	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+110	120	propeptide	structure_element	(d) Structural superposition of the matured β2 active site, the WT β2-T1A propeptide and the β2-T(-2)V mutant propeptide.	FIG
+9	16	Val(-2)	residue_name_number	Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1).	FIG
+51	60	S1 pocket	site	Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1).	FIG
+98	105	Gly(-1)	residue_name_number	Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1).	FIG
+144	148	Thr1	residue_name_number	Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1).	FIG
+182	189	Gly(-1)	residue_name_number	Notably, Val(-2) of the latter does not occupy the S1 pocket, thereby changing the orientation of Gly(-1) and preventing nucleophilic attack of Thr1Oγ on the carbonyl carbon atom of Gly(-1).	FIG
+64	75	active site	site	Architecture and proposed reaction mechanism of the proteasomal active site.	FIG
+4	28	Hydrogen-bonding network	site	(a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines).	FIG
+36	42	mature	protein_state	(a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines).	FIG
+43	45	WT	protein_state	(a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines).	FIG
+46	48	β5	protein	(a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines).	FIG
+61	72	active site	site	(a) Hydrogen-bonding network at the mature WT β5 proteasomal active site (dotted lines).	FIG
+0	4	Thr1	residue_name_number	Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ.	FIG
+29	34	Lys33	residue_name_number	Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ.	FIG
+76	81	Asp17	residue_name_number	Thr1OH is hydrogen-bonded to Lys33NH2 (2.7 Å), which in turn interacts with Asp17Oδ.	FIG
+4	8	Thr1	residue_name_number	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+54	60	Ser129	residue_name_number	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+95	98	168	residue_number	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+100	106	Ser169	residue_name_number	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+113	119	Asp166	residue_name_number	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+151	171	active-site residues	site	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+205	223	strictly conserved	protein_state	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+232	250	proteolytic centre	site	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+264	277	superposition	experimental_method	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+285	295	β subunits	protein	The Thr1 N terminus is engaged in hydrogen bonds with Ser129Oγ, the carbonyl oxygen of residue 168, Ser169Oγ and Asp166Oδ. (b) The orientations of the active-site residues involved in hydrogen bonding are strictly conserved in each proteolytic centre, as shown by superposition of the β subunits.	FIG
+4	28	Structural superposition	experimental_method	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+36	38	WT	protein_state	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+39	41	β5	protein	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+50	57	β5-K33A	mutant	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+58	60	pp	chemical	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+61	66	trans	protein_state	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+67	73	mutant	protein_state	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+74	85	active site	site	(c) Structural superposition of the WT β5 and the β5-K33A pp trans mutant active site.	FIG
+17	22	water	chemical	In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located.	FIG
+83	85	WT	protein_state	In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located.	FIG
+126	131	Lys33	residue_name_number	In the latter, a water molecule (red sphere) is found at the position where in the WT structure the side chain amine group of Lys33 is located.	FIG
+13	18	Lys33	residue_name_number	Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH.	FIG
+24	29	water	chemical	Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH.	FIG
+57	62	Arg19	residue_name_number	Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH.	FIG
+65	70	Asp17	residue_name_number	Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH.	FIG
+77	81	Thr1	residue_name_number	Similarly to Lys33, the water molecule hydrogen bonds to Arg19O, Asp17Oδ and Thr1OH.	FIG
+38	43	water	chemical	Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place.	FIG
+77	81	Thr1	residue_name_number	Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place.	FIG
+99	119	active-site residues	site	Note, the strong interaction with the water molecule causes a minor shift of Thr1, while all other active-site residues remain in place.	FIG
+45	79	autocatalytic precursor processing	ptm	(d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome.	FIG
+103	113	proteasome	complex_assembly	(d) Proposed chemical reaction mechanism for autocatalytic precursor processing and proteolysis in the proteasome.	FIG
+4	15	active-site	site	The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red.	FIG
+16	20	Thr1	residue_name_number	The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red.	FIG
+46	56	propeptide	structure_element	The active-site Thr1 is depicted in blue, the propeptide segment and the peptide substrate are coloured in green, whereas the scissile peptide bond is highlighted in red.	FIG
+0	9	Autolysis	ptm	Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state.	FIG
+68	72	Thr1	residue_name_number	Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state.	FIG
+79	84	Lys33	residue_name_number	Autolysis (left set of structures) is initiated by deprotonation of Thr1OH via Lys33NH2 and the formation of a tetrahedral transition state.	FIG
+4	22	strictly conserved	protein_state	The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle.	FIG
+37	42	Gly47	residue_name_number	The strictly conserved oxyanion hole Gly47NH stabilizing the negatively charged intermediate is illustrated as a semicircle.	FIG
+43	47	Thr1	residue_name_number	Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH.	FIG
+102	112	propeptide	structure_element	Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH.	FIG
+151	157	Asp166	residue_name_number	Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH.	FIG
+164	170	Ser129	residue_name_number	Collapse of the transition state frees the Thr1 N terminus (by completing an N-to-O acyl shift of the propeptide), which is subsequently protonated by Asp166OH via Ser129OH.	FIG
+6	10	Thr1	residue_name_number	Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate.	FIG
+26	31	water	chemical	Next, Thr1NH2 polarizes a water molecule for the nucleophilic attack of the acyl-enzyme intermediate.	FIG
+33	44	active-site	site	On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures).	FIG
+45	49	Thr1	residue_name_number	On hydrolysis of the latter, the active-site Thr1 is ready for catalysis (right set of structures).	FIG
+12	16	Thr1	residue_name_number	The charged Thr1 N terminus may engage in the orientation of the amide moiety and donate a proton to the emerging N terminus of the C-terminal cleavage product.	FIG
+27	31	Thr1	residue_name_number	The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme.	FIG
+55	60	water	chemical	The resulting deprotonated Thr1NH2 finally activates a water molecule for hydrolysis of the acyl-enzyme.	FIG
+4	14	proteasome	complex_assembly	The proteasome favours threonine as the active-site nucleophile.	FIG
+23	32	threonine	residue_name	The proteasome favours threonine as the active-site nucleophile.	FIG
+4	35	Growth tests by serial dilution	experimental_method	(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.	FIG
+39	41	WT	protein_state	(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.	FIG
+52	54	β5	protein	(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.	FIG
+56	62	mutant	protein_state	(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.	FIG
+63	68	yeast	taxonomy_domain	(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.	FIG
+107	118	active-site	site	(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.	FIG
+119	126	mutants	experimental_method	(a) Growth tests by serial dilution of WT and pre2 (β5) mutant yeast cultures reveal growth defects of the active-site mutants under the indicated conditions after 2 days (2 d) of incubation.	FIG
+13	15	WT	protein_state	(b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC.	FIG
+20	26	mutant	protein_state	(b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC.	FIG
+27	38	proteasomes	complex_assembly	(b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC.	FIG
+89	91	β5	protein	(b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC.	FIG
+113	125	Suc-LLVY-AMC	chemical	(b) Purified WT and mutant proteasomes were tested for their chymotrypsin-like activity (β5) using the substrate Suc-LLVY-AMC.	FIG
+24	51	2FO–FC electron-density map	evidence	(c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment.	FIG
+88	94	β5-T1C	mutant	(c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment.	FIG
+95	105	propeptide	structure_element	(c) Illustration of the 2FO–FC electron-density map (blue mesh contoured at 1σ) for the β5-T1C propeptide fragment.	FIG
+4	14	prosegment	structure_element	The prosegment is cleaved but still bound in the substrate-binding channel.	FIG
+18	25	cleaved	protein_state	The prosegment is cleaved but still bound in the substrate-binding channel.	FIG
+30	41	still bound	protein_state	The prosegment is cleaved but still bound in the substrate-binding channel.	FIG
+49	74	substrate-binding channel	site	The prosegment is cleaved but still bound in the substrate-binding channel.	FIG
+9	16	His(-2)	residue_name_number	Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant.	FIG
+37	46	S1 pocket	site	Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant.	FIG
+57	62	Met45	residue_name_number	Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant.	FIG
+101	112	β5-T1A-K81R	mutant	Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant.	FIG
+113	119	mutant	protein_state	Notably, His(-2) does not occupy the S1 pocket formed by Met45, similar to what was observed for the β5-T1A-K81R mutant.	FIG
+4	28	Structural superposition	experimental_method	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+36	47	β5-T1A-K81R	mutant	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+56	62	β5-T1C	mutant	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+63	69	mutant	protein_state	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+88	90	WT	protein_state	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+91	93	β5	protein	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+107	131	Structural superposition	experimental_method	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+139	145	β5-T1C	mutant	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+146	156	propeptide	structure_element	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+166	172	β1-T1A	mutant	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+173	184	active site	site	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+200	202	WT	protein_state	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+203	205	β5	protein	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+206	217	active site	site	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+218	233	in complex with	protein_state	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+238	248	proteasome	complex_assembly	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+259	264	MG132	chemical	(d) Structural superposition of the β5-T1A-K81R and the β5-T1C mutant subunits onto the WT β5 subunit. (e) Structural superposition of the β5-T1C propeptide onto the β1-T1A active site (blue) and the WT β5 active site in complex with the proteasome inhibitor MG132 (ref.).	FIG
+4	13	inhibitor	chemical	The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel.	FIG
+29	40	propeptides	structure_element	The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel.	FIG
+76	101	substrate-binding channel	site	The inhibitor as well as the propeptides adopt similar conformations in the substrate-binding channel.	FIG
+4	28	Structural superposition	experimental_method	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+36	38	WT	protein_state	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+39	41	β5	protein	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+46	52	β5-T1C	mutant	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+53	59	mutant	protein_state	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+60	72	active sites	site	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+137	141	Thr1	residue_name_number	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+170	174	Cys1	residue_name_number	(f) Structural superposition of the WT β5 and β5-T1C mutant active sites illustrates the different orientations of the hydroxyl group of Thr1 and the thiol side chain of Cys1.	FIG
+4	28	Structural superposition	experimental_method	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+36	38	WT	protein_state	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+39	41	β5	protein	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+46	52	β5-T1S	mutant	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+53	59	mutant	protein_state	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+60	72	active sites	site	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+130	134	Thr1	residue_name_number	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+139	143	Ser1	residue_name_number	(g) Structural superposition of the WT β5 and β5-T1S mutant active sites reveals different orientations of the hydroxyl groups of Thr1 and Ser1, respectively.	FIG
+4	31	2FO–FC electron-density map	evidence	The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated.	FIG
+36	40	Ser1	residue_name_number	The 2FO–FC electron-density map for Ser1 (blue mesh contoured at 1σ) is illustrated.	FIG
+24	28	Thr1	residue_name_number	(h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ.	FIG
+74	79	Ala46	residue_name_number	(h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ.	FIG
+86	90	Thr3	residue_name_number	(h) The methyl group of Thr1 is anchored by hydrophobic interactions with Ala46Cβ and Thr3Cγ.	FIG
+0	4	Ser1	residue_name_number	Ser1 lacks this stabilization and is therefore rotated by 60°.	FIG
+5	10	lacks	protein_state	Ser1 lacks this stabilization and is therefore rotated by 60°.	FIG
+14	16	WT	protein_state	Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib.	FIG
+21	27	mutant	protein_state	Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib.	FIG
+28	34	β5-T1S	mutant	Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib.	FIG
+35	46	proteasomes	complex_assembly	Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib.	FIG
+50	60	bortezomib	chemical	Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib.	FIG
+65	76	carfilzomib	chemical	Inhibition of WT and mutant β5-T1S proteasomes by bortezomib and carfilzomib.	FIG
+0	17	Inhibition assays	experimental_method	Inhibition assays (left panel).	FIG
+9	14	yeast	taxonomy_domain	Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.	FIG
+15	26	proteasomes	complex_assembly	Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.	FIG
+78	80	β5	protein	Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.	FIG
+108	118	bortezomib	chemical	Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.	FIG
+123	134	carfilzomib	chemical	Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.	FIG
+155	167	Suc-LLVY-AMC	chemical	Purified yeast proteasomes were tested for the susceptibility of their ChT-L (β5) activity to inhibition by bortezomib and carfilzomib using the substrate Suc-LLVY-AMC.	FIG
+0	11	IC50 values	evidence	IC50 values were determined in triplicate; s.d.'s are indicated by error bars.	FIG
+10	21	IC50 values	evidence	Note that IC50 values depend on time and enzyme concentration.	FIG
+0	11	Proteasomes	complex_assembly	Proteasomes (final concentration: 66 nM) were incubated with inhibitor for 45 min before substrate addition (final concentration: 200 μM).	FIG
+0	10	Structures	evidence	Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel).	FIG
+18	24	β5-T1S	mutant	Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel).	FIG
+25	31	mutant	protein_state	Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel).	FIG
+35	60	complex with both ligands	complex_assembly	Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel).	FIG
+93	97	Ser1	residue_name_number	Structures of the β5-T1S mutant in complex with both ligands (green) prove the reactivity of Ser1 (right panel).	FIG
+4	32	2FO–FC electron-density maps	evidence	The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ.	FIG
+49	53	Ser1	residue_name_number	The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ.	FIG
+112	119	P1 site	site	The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ.	FIG
+121	125	Leu1	residue_name_number	The 2FO–FC electron-density maps (blue mesh) for Ser1 (brown) and the covalently bound ligands (green; only the P1 site (Leu1) is shown) are contoured at 1σ.	FIG
+4	6	WT	protein_state	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+7	35	proteasome:inhibitor complex	complex_assembly	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+36	46	structures	evidence	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+67	71	Thr1	residue_name_number	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+86	98	superimposed	experimental_method	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+120	128	mutation	experimental_method	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+132	136	Thr1	residue_name_number	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+140	143	Ser	residue_name	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+180	190	bortezomib	chemical	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG
+194	205	carfilzomib	chemical	The WT proteasome:inhibitor complex structures (inhibitor in grey; Thr1 in black) are superimposed and demonstrate that mutation of Thr1 to Ser does not affect the binding mode of bortezomib or carfilzomib.	FIG