PDB-7uyi: Crystal structure of the computationally optimized broadly reacti...

Basic information

• Entry: Database: PDB / ID: 7uyi
• Title: Crystal structure of the computationally optimized broadly reactive H1 influenza hemagglutinin P1
• Components: COBRA P1 HA
• Keywords: VIRAL PROTEIN / hemagglutinin / virus entry / antigenic
• Biological species: Influenza A virus
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
• Authors: Dzimianski, J.V. / DuBois, R.M.

Funding support

United States, 1items

Organization	Grant number	Country
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)	75N93019C00052	United States

• Citation: Journal: Commun Biol / Year: 2023; Title: Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine.; Authors: John V Dzimianski / Julianna Han / Giuseppe A Sautto / Sara M O'Rourke / Joseph M Cruz / Spencer R Pierce / Jeffrey W Ecker / Michael A Carlock / Kaito A Nagashima / Jarrod J Mousa / Ted M Ross / Andrew B Ward / Rebecca M DuBois / ; Abstract: Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding.

History

Deposition	May 6, 2022	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Apr 19, 2023	Provider: repository / Type: Initial release
Revision 1.1	May 3, 2023	Group: Database references / Category: citation / citation_author Item: _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2	May 1, 2024	Group: Data collection / Database references Category: chem_comp_atom / chem_comp_bond / citation / citation_author Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

Assembly

Deposited unit

A: COBRA P1 HA

B: COBRA P1 HA

C: COBRA P1 HA

E: COBRA P1 HA

F: COBRA P1 HA

D: COBRA P1 HA

hetero molecules

Summary:

• 356 kDa, 6 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	356,483	37
Polymers	345,079	6
Non-polymers	11,404	31
Water	414	23

1

A: COBRA P1 HA

C: COBRA P1 HA

E: COBRA P1 HA

hetero molecules

Summary:

• defined by author
• Evidence: gel filtration
• 178 kDa, 3 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	178,050	18
Polymers	172,540	3
Non-polymers	5,510	15
Water	54	3

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

2

B: COBRA P1 HA

F: COBRA P1 HA

D: COBRA P1 HA

hetero molecules

Summary:

• defined by author
• Evidence: gel filtration
• 178 kDa, 3 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	178,433	19
Polymers	172,540	3
Non-polymers	5,893	16
Water	54	3

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Unit cell

Length a, b, c (Å)	264.849, 77.560, 222.523
Angle α, β, γ (deg.)	90.000, 93.773, 90.000
Int Tables number	5
Space group name H-M	C121
Space group name Hall	C2y
Symmetry operation	#1: x,y,z #2: -x,y,-z #3: x+1/2,y+1/2,z #4: -x+1/2,y+1/2,-z

Components

Protein / Non-polymers , 2 types, 29 molecules A B C E F D

#1: Protein

A B C E F D
COBRA P1 HA / Hemagglutinin P1

Details:

Mass: 57513.211 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)

#7: Water

ChemComp-HOH / water / Water

Details:

Mass: 18.015 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Formula: H₂O

Sugars , 5 types, 31 molecules

#2: Polysaccharide

A - [G] F - [P] alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose

Details:

Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source

Descriptor:

Descriptor	Type	Program
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROH	Glycam Condensed Sequence	GMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1	WURCS	PDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}	LINUCS	PDB-CARE

#3: Polysaccharide

A - [H] A - [I] B - [K] E - [O] F - [Q]
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose

Details:

Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source

Descriptor:

Descriptor	Type	Program
DGlcpNAcb1-4DGlcpNAcb1-ROH	Glycam Condensed Sequence	GMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1	WURCS	PDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}	LINUCS	PDB-CARE

#4: Polysaccharide

B - [J] B - [L] C - [M] beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose

Details:

Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source

Descriptor:

Descriptor	Type	Program
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROH	Glycam Condensed Sequence	GMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1	WURCS	PDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}	LINUCS	PDB-CARE

#5: Polysaccharide

E - [N] D - [R] alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose

Details:

Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source

Descriptor:

Descriptor	Type	Program
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROH	Glycam Condensed Sequence	GMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1	WURCS	PDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}	LINUCS	PDB-CARE

#6: Sugar

A-601 A-602 A-603 A-604 B-601 B-602 B-603 B-604 C-601 C-602 C-603 C-604 E-601 F-601 F-602 D-601 D-602 D-603 D-604
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine

Details:

Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 19 / Source method: obtained synthetically / Formula: C₈H₁₅NO₆

Identifier:

Identifier	Type	Program
DGlcpNAcb	CONDENSED IUPAC CARBOHYDRATE SYMBOL	GMML 1.0
N-acetyl-b-D-glucopyranosamine	COMMON NAME	GMML 1.0
b-D-GlcpNAc	IUPAC CARBOHYDRATE SYMBOL	PDB-CARE 1.0
GlcNAc	SNFG CARBOHYDRATE SYMBOL	GMML 1.0

Details

• Has ligand of interest: N

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 3.3 ų/Da / Density % sol: 62.78 % / Description: Plates
• Crystal grow: Temperature: 295 K / Method: vapor diffusion, hanging drop / pH: 8.5 ; Details: 0.25 M magnesium chloride, 0.1 M Tris-HCl, pH 8.5, 15% PEG4000, 6% PEG400

Data collection

• Diffraction: Mean temperature: 100 K / Serial crystal experiment: N
• Diffraction source: Source: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1.03 Å
• Detector: Type: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 21, 2020
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1.03 Å / Relative weight: 1
• Reflection: Resolution: 3→50 Å / Num. obs: 90769 / % possible obs: 99.8 % / Redundancy: 6.2 % / B_iso Wilson estimate: 63 Ų / CC_1/2: 0.988 / R_merge(I) obs: 0.222 / Net I/σ(I): 6.6
• Reflection shell: Resolution: 3→3.05 Å / R_merge(I) obs: 1.435 / Mean I/σ(I) obs: 1.7 / Num. unique obs: 4343 / CC_1/2: 0.429 / % possible all: 97.7

Processing

Software

Name	Version	Classification
Blu-Ice		data collection
DIALS		data reduction
Aimless		data scaling
PHASER		phasing
PHENIX	1.19.2_4158	refinement
Coot		model building

Refinement

Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 3→47.75 Å / SU ML: 0.3911 / Cross valid method: FREE R-VALUE / σ(F): 1.91 / Phase error: 26.0034
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	Rfactor	Num. reflection	% reflection
Rfree	0.2698	4570	5.04 %
Rwork	0.2258	86181	-
obs	0.2281	90750	99.81 %

• Solvent computation: Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
• Displacement parameters: B_iso mean: 80.54 Ų

Refinement step

Cycle: LAST / Resolution: 3→47.75 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	22556	0	745	23	23324

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0022	23834
X-RAY DIFFRACTION	f_angle_d	0.4783	32297
X-RAY DIFFRACTION	f_chiral_restr	0.0407	3635
X-RAY DIFFRACTION	f_plane_restr	0.0035	4107
X-RAY DIFFRACTION	f_dihedral_angle_d	11.0929	8814

LS refinement shell

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Refine-ID	% reflection obs (%)
3-3.03	0.3583	137	0.3101	2709	X-RAY DIFFRACTION	96.64
3.03-3.07	0.2892	145	0.276	2907	X-RAY DIFFRACTION	100
3.07-3.11	0.3313	172	0.2689	2829	X-RAY DIFFRACTION	100
3.11-3.15	0.3049	149	0.2639	2852	X-RAY DIFFRACTION	100
3.15-3.19	0.3074	131	0.2643	2865	X-RAY DIFFRACTION	100
3.19-3.23	0.3151	162	0.2662	2894	X-RAY DIFFRACTION	99.93
3.23-3.28	0.3355	161	0.2675	2826	X-RAY DIFFRACTION	100
3.28-3.33	0.2844	153	0.2609	2836	X-RAY DIFFRACTION	99.97
3.33-3.38	0.3014	147	0.2537	2892	X-RAY DIFFRACTION	99.9
3.38-3.43	0.2788	157	0.2624	2798	X-RAY DIFFRACTION	99.97
3.43-3.49	0.2809	151	0.2439	2880	X-RAY DIFFRACTION	100
3.49-3.56	0.277	174	0.2437	2841	X-RAY DIFFRACTION	99.97
3.56-3.63	0.3013	150	0.2386	2858	X-RAY DIFFRACTION	99.97
3.63-3.7	0.2974	178	0.2415	2865	X-RAY DIFFRACTION	100
3.7-3.78	0.2743	154	0.2378	2834	X-RAY DIFFRACTION	100
3.78-3.87	0.2778	143	0.2468	2885	X-RAY DIFFRACTION	99.87
3.87-3.96	0.3101	129	0.231	2883	X-RAY DIFFRACTION	99.97
3.96-4.07	0.2613	133	0.2141	2914	X-RAY DIFFRACTION	99.97
4.07-4.19	0.2563	137	0.2095	2894	X-RAY DIFFRACTION	99.93
4.19-4.33	0.2444	152	0.2073	2842	X-RAY DIFFRACTION	99.87
4.33-4.48	0.2393	144	0.2007	2878	X-RAY DIFFRACTION	100
4.48-4.66	0.2815	144	0.1922	2879	X-RAY DIFFRACTION	99.97
4.66-4.87	0.2499	156	0.1892	2905	X-RAY DIFFRACTION	99.93
4.87-5.13	0.2095	133	0.1944	2907	X-RAY DIFFRACTION	99.84
5.13-5.45	0.2387	200	0.2034	2822	X-RAY DIFFRACTION	99.8
5.45-5.87	0.24	151	0.2014	2917	X-RAY DIFFRACTION	100
5.87-6.46	0.2439	158	0.2181	2902	X-RAY DIFFRACTION	99.87
6.46-7.39	0.256	168	0.2167	2886	X-RAY DIFFRACTION	99.8
7.39-9.3	0.2377	148	0.2202	2953	X-RAY DIFFRACTION	99.77
9.3-47.75	0.2891	153	0.2291	3028	X-RAY DIFFRACTION	99.25

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°2)	L12 (°2)	L13 (°2)	L22 (°2)	L23 (°2)	L33 (°2)	S11 (Å °)	S12 (Å °)	S13 (Å °)	S21 (Å °)	S22 (Å °)	S23 (Å °)	S31 (Å °)	S32 (Å °)	S33 (Å °)	T11 (Å2)	T12 (Å2)	T13 (Å2)	T22 (Å2)	T23 (Å2)	T33 (Å2)	Origin x (Å)	Origin y (Å)	Origin z (Å)
1	5.16482814017	-1.25354318655	0.283528886188	1.07866260979	0.175746104719	0.964221440569	-0.00583200148841	-0.374347363175	-0.927096203011	0.210754762944	0.015508638778	0.0559393540874	-0.0190171739649	0.292282819136	0.0151319767066	0.351278049648	-0.0720565203987	-0.0689953167536	0.399883363357	0.117185795982	0.412681892248	-61.154	5.46	105.595
2	2.46313194322	-0.342878685996	0.944898256886	3.259107783	-0.111832837276	2.78924093408	-0.0295834134727	-1.29391415638	-0.41103364227	0.518942612619	0.00377965681469	0.545725203574	-0.23178576068	-0.804335391062	-0.0326553179942	0.400398036765	0.0364180982405	0.103818716267	0.541101354526	0.0558860008314	0.341907378847	-94.841	20.181	109.826
3	1.6342218102	1.13887198531	-1.38934968337	8.71155715322	-3.69350373693	4.91033429011	0.169715227814	-0.269105822346	0.432501090414	-0.395928129191	0.291542407227	1.71634249971	-0.75907811432	-0.958177776413	-0.179579635689	0.518410753528	0.013828707891	0.0186499250324	0.716407172568	-0.0210949613863	0.281326547765	-101.885	20.081	97.733
4	1.75901624165	-1.14074187131	0.826101370119	2.53079338205	-0.192698494311	2.10685748241	-0.127894888617	-0.612698464015	-0.301218826881	0.348281574773	0.112567530688	0.145178275159	-0.0625489209723	-0.389631307111	0.0701791402943	0.381877865789	-0.0739774087963	0.0401737989646	0.390784644994	0.104278949022	0.308463352849	-88.147	16.258	103.93
5	1.73642629059	-1.3416936991	0.322503548952	1.86064542859	-0.339740559201	0.986956361	0.236015470086	-0.700331533278	-0.708414865808	0.45015482036	-0.13753439886	-0.700932566146	-0.17882309885	0.561769423062	-0.0476468169269	0.293069004221	-0.0544813901428	-0.211680029784	0.512958474098	0.0971015417034	0.627926191455	-50.952	4.528	102.969
6	2.39383491028	-2.00298889377	0.623067301062	1.64498204621	-0.595321030776	2.15943719956	0.165805359935	0.240579849636	-0.442030275212	0.170309911944	-0.149807274011	-0.403454649492	-0.284529565471	0.618816280653	0.172053302576	0.291070115313	-0.0959863048374	-0.0414709874507	0.992881850616	-0.0666372425835	1.2520745541	-18.572	1.281	101.352
7	8.6258820697	-1.19338752827	-3.89673199441	0.405526746105	1.59804458164	6.48080046348	0.964856811761	1.46429592034	1.22681534862	0.0362222250897	0.301248162888	0.412425596843	-0.506085468023	-0.633742524387	-0.965464261505	0.211129846913	0.0368131422855	-0.00753236188109	0.801799219942	0.298987613378	1.09243454942	-47.324	13.324	95.467
8	1.50330566027	0.346061522391	0.0366875656322	0.122868878821	-0.108216262665	1.87126240252	0.0147654067172	-0.0403191820353	-0.603767572541	0.0409929790492	0.0306532057606	0.140708961645	0.450920115858	0.764418733682	-0.122545617538	0.274976039969	0.0562586109531	-0.0440913959134	0.320048435072	-0.114848850462	0.656214758493	-60.81	5.397	89.386
9	3.93931222284	-1.09721911182	-2.83315363973	6.06889540287	1.1907033776	2.05920436958	0.245120989452	1.41936787107	-0.0377088624453	-0.492532061997	-0.379855430042	-1.11077860627	-0.618974289568	1.21476848848	0.160202649837	0.524912564695	-0.125977393527	-0.0492761199957	1.39864071386	0.00917476058943	0.816795791446	-14.619	2.575	90.088
10	2.38713695061	-0.605951207227	1.64504640799	2.6246588151	0.615108171575	1.62688319725	0.413205005674	-0.263954584881	0.347674229344	-0.117531801967	0.0269738036293	-0.947359545893	-0.671069511562	0.977000501226	-0.00278274491057	0.416710114245	-0.168892596781	-0.0324153330722	2.0170688244	-0.208219434484	1.49889777509	0.33	0.431	96.607
11	5.11395582889	0.850601473984	-1.44634912759	1.46164114995	0.0192164415668	0.496076986226	-0.0672318725598	-0.0483857625024	1.05712176232	-0.222200881142	0.05271597205	0.0505961327736	-0.0568823084729	0.225207236634	-0.0877304376922	0.418364881805	0.0549456519739	0.162747725119	0.55225293937	0.186400957826	0.366076018519	-47.027	-22.113	4.421
12	2.28295813508	0.717482031801	-1.490990561	2.6378635285	0.850851420088	3.00134076748	-0.197166572664	0.963028390979	0.398246758425	-0.528161758994	0.174840413322	0.735811626067	-8.16687203783E-5	-0.729745484993	0.005577321801	0.480408910111	0.0516824104984	-0.119656081606	0.554692437831	0.10617904967	0.377996768403	-86.106	-35.159	2.984
13	3.06825723031	-1.50989295598	2.42481464326	5.28296422831	-2.2238558733	7.94179361459	-0.357908734771	-0.556822623701	-0.0956950183681	1.4629179157	0.460414572058	0.598506282361	0.623334357703	-0.765095808094	-0.141994657161	0.577565479353	-0.0544747916961	0.0672206189527	0.380904288534	-0.0977231505027	0.330178749734	-91.357	-40.834	15.501
14	4.40754536888	1.560478189	-2.43254757094	0.702654364698	-0.33671707194	3.17928150776	-0.0228155371683	-0.611271587558	1.03277199829	0.343067703162	0.653122802577	0.151683847828	-0.0170849119194	-0.483366117814	-0.0526621272684	0.394740829507	0.250831769925	-0.0594381930695	0.955239720378	0.333107281683	1.0002580407	-96.968	-23.142	15.193
15	2.54667907906	0.873419159521	-0.926847072831	1.51095998155	-0.753428270327	2.89679629522	-0.191517585841	0.607130168824	0.501916275933	-0.347609660204	0.0949325433582	0.14971162774	0.173949339799	-0.396900463429	0.136274748271	0.444877991377	0.0286072072137	-0.0441436467139	0.305818854214	0.0657644590957	0.312076498489	-82.443	-36.875	7.305
16	2.98511909034	2.66553999399	0.0613201051728	3.01529151891	0.096917487885	1.94004846084	0.342190293472	0.218980274726	0.00185723447213	-0.266711458116	-0.275783328004	-0.637575255077	0.381224824849	0.4159154333	-0.065916521673	0.356889353055	0.107594759923	0.215454047704	0.55629552652	0.127899242191	0.683715785731	-43.772	-21.365	7.68
17	0.0565288263889	-0.0123851220256	0.109039334943	0.377874447057	0.352094280449	0.659554023028	0.327524306535	0.357831843196	0.434762562374	-0.295503817439	0.148160494739	-0.345487421102	0.00464305785349	0.820617890244	0.800417893944	-0.00968810298937	-0.0787314338764	0.267191994916	1.73873869737	-0.279263080447	1.47325134953	-7.059	-18.028	7.526
18	9.81782318961	-0.47087700285	-4.23854731851	8.30453645219	-0.92754622185	6.08620629654	0.759771048972	-0.738813396582	-1.36687288533	-0.368913263679	-0.523700765135	-0.428756954062	0.412165745104	0.613061553069	-0.118872653587	0.399432491937	0.0902321587692	0.0249935770013	0.696699641901	-0.013376529554	0.662911702142	-30.412	-29.867	14.907
19	2.47062755063	0.632635601051	-1.19264085834	0.959583619393	-0.0180569539049	0.838761475678	0.40230785776	-0.75375626235	0.360090107846	0.0168791840518	-0.0940094301693	-0.216624113082	-0.0458305223429	0.914491278132	-0.0646916196629	0.322229994101	-0.0141662659026	0.0845936488082	0.940396020121	-0.0864609176213	0.538335090795	-27.496	-20.615	19.55
20	1.44096804777	1.91562531347	-2.48885402883	3.92299359928	-3.87673802178	4.53403524899	0.559854512456	-0.892664623915	-1.61210490012	0.281074032589	-0.625800748275	-1.66814953297	0.620987043489	1.95283802493	0.0360323807789	0.668296109099	0.396094766904	-0.128712600924	1.76772374035	-0.0775846851842	1.22214994038	9.294	-22.812	14.434
21	2.85659240559	0.712151134623	0.0467200193242	1.09213527308	0.178486781414	0.760576085493	-0.0904789716797	0.272212757376	1.20175695786	-0.239998947035	-0.177177482638	-0.89940529257	-1.01422345849	0.503644648885	-0.0843294804019	0.267788790859	-0.555080432625	0.0258032565314	0.737803281688	0.199117561519	0.652227907241	-51.666	31.1	75.541
22	4.04055598366	0.857973044196	-0.732613927948	3.15011856467	-0.425468811639	3.65036517807	-0.177739031991	0.21446421578	1.17749976237	-0.215825308184	0.123852056548	0.710380919968	-0.682503214421	-0.241036406595	0.0526818159549	0.53159173429	0.00543262697082	-0.161914450341	0.278616444994	0.0149201769901	0.482106769927	-92.394	34.418	73.351
23	8.50949661271	-2.88939304714	-1.34785100172	6.05076240907	2.47406594932	2.41171737403	0.13672009666	0.459319148062	-0.87327919343	0.146336144849	-0.595487221609	1.40076823277	0.398725073547	-0.496578339032	0.476154410311	0.354785035758	-0.00785355015447	-0.074157996799	0.382657550931	0.0604506132103	0.668541311835	-100.743	23.671	77.292
24	3.55597069375	0.972408439305	1.02190778553	2.44528812865	0.0365583653256	0.445430422217	-0.0239226206443	-0.103654184169	0.95272439607	-0.0265620184945	-0.0389118024214	0.549062020913	0.0216608553861	0.0862021316524	0.091904043707	0.370572281663	-0.043572988171	-0.00658190099116	0.345203022851	-0.108927992352	0.424082406194	-90.583	29.535	75.149
25	3.61286914784	-0.664968146316	-0.67199309278	1.58658229336	0.722344954641	1.36167730588	-0.106452936175	0.455794930054	0.88376786431	-0.148247060747	-0.176110534205	-0.931842753964	-0.246448692876	0.357554128273	0.0490148524156	0.428372457847	-0.268463234496	0.102549153625	0.732491972074	0.177589606422	0.499795733128	-49.914	28.549	76.552
26	1.1185917587	0.171689557225	0.216558240921	0.0256299406704	0.0424849939944	0.0389728701954	-0.104585805222	0.128387900197	0.0150550353639	-0.0911601998589	-0.160718202242	-0.495429134615	-0.230683135945	0.791260184373	-0.407665796878	0.101782550695	-0.160816680676	0.328557989777	1.90408865859	-0.0980075611885	2.23005645172	-14.525	21.769	74.911
27	5.36558911419	-4.38301532783	0.226987420266	7.33202560568	2.92537258217	2.69759052554	-1.09798498669	0.520565719062	-3.78958095964	0.590970267201	-0.519066125045	-0.654356286294	0.425142355456	0.571313544194	1.49237765499	1.08474046421	-0.420555689317	0.505699353269	1.6895436697	0.24268559438	1.63724639574	-2.043	23.895	69.059
28	0.173853749484	-0.785629408531	0.165403463239	4.48452131346	0.373181802121	1.37768522357	-0.13044192011	1.24939563052	-1.4779955522	-0.0587844815126	-0.421156375741	-0.548474414775	0.0662346925854	0.689173677026	0.396510232608	0.605998872303	-0.0376932811553	0.181051203966	1.2799971435	0.16645368554	0.708983667321	-37.505	16.316	69.376
29	0.79470399635	0.664947212865	0.239551815238	0.945422188044	-0.577219386006	0.92294778871	-0.0472403797343	0.227152871743	-0.231837272821	-0.292761302738	-0.17141691343	-0.863257610873	-0.00234665212546	1.05702088454	-0.345174730826	0.371289875486	-0.0637498433244	0.127892812682	1.34038975704	0.0988865896312	1.06129819711	-29.779	13.413	78.015
30	4.88080309722	0.909072714192	3.7674404483	2.18593268642	-0.50251731927	3.62781885838	0.104893143523	0.0233883730541	-1.00696477205	0.237068922776	0.862344261078	1.04739500592	-0.219691919691	-2.37031835142	-0.481633993588	0.794081825988	0.0770502692515	0.699214416232	2.53312688763	-0.234507065126	2.57989791804	5.251	6.208	64.392
31	0.891470018621	-0.132056948574	-0.0192356210456	0.0771149474285	0.173145983425	0.229251834546	-0.0875065923531	-0.114506612675	-1.19017059374	0.200326909959	-0.0516590409124	-0.953748996795	0.720183755606	0.726619245621	0.0209078746247	0.71803906443	0.481084823195	-0.0619419125539	1.06398364003	0.155116869256	1.07678034498	-41.57	-47.797	34.046
32	9.69173193809	-0.865585302513	4.4175349305	0.994623209992	0.00746411998105	3.48896525446	-0.245081123758	0.351840950311	-0.465687805399	0.184020400829	-0.131475135288	0.0565908851792	0.307619617678	-0.066976771534	0.290708304207	0.786788909363	0.0030703595248	0.161577784565	0.311980188221	0.0678870493614	0.314382793081	-70.057	-55.108	35.91
33	3.333339655	-0.840431946047	-0.300391560251	2.52017500876	-0.425134493753	2.74581521377	-0.00519263458326	-0.191359160954	-0.442334651514	0.655653612609	-0.235670456227	0.782810186304	0.723315807488	-0.171310869364	0.170492468547	0.799939440014	-0.119397034416	0.239824704127	0.24557893469	-0.0800055209099	0.289911861998	-86.328	-48.876	36.949
34	4.3701322741	2.88362947284	-1.05419178473	4.07321379636	1.38496647896	2.17030092048	0.372467469724	-0.935025554253	-0.738122372522	0.44842244332	-0.290320221002	-0.893893368598	0.614766774138	0.476719474883	0.0651728963853	0.657221910104	0.319687870483	-0.119280152682	0.772808667034	0.173047501516	0.610515116344	-45.165	-46.717	35.348
35	0.304214410536	0.729373648444	0.310411353262	1.76105949519	0.759764634025	0.33135246046	-0.353640106155	0.16979640022	-0.132128990575	0.620847001354	0.399113048783	-0.134338366194	0.638445094742	0.413181472203	0.113028884866	0.431133504546	0.297427063932	0.104761376439	1.35138492359	0.0974683700707	1.76996301496	-6.749	-39.509	36.721
36	5.86918837705	7.53250661088	5.82058806733	9.98024792462	7.18625603627	6.03460285765	-0.550951742375	-2.0696433968	0.490467051629	0.106051417027	-0.875686238266	1.57892213972	0.995216799407	-0.0720290966983	1.29035438497	0.737050282544	-0.150862025628	-0.212063865834	1.52398304067	0.129283036716	1.01956072332	-10.863	-34.553	45.718
37	7.2466758599	5.28566272215	2.40912844194	4.42352233825	0.950329827933	7.94138680941	1.37391182038	-0.57834071412	2.13751957144	0.652822078261	-1.34830714824	-0.0619467649619	-0.270675302495	-0.364242449166	0.0456236824885	0.685649568082	0.142141841968	-0.232864306353	0.8484030988	-0.0892343430997	1.08223104986	-33.452	-32.979	41.119
38	7.97713275814	0.0939855547706	3.65584518582	2.42313674083	0.704309679036	3.02780508964	-0.182818173777	-0.236505635677	0.441997256684	-0.101952990408	-0.0279359905197	0.146949432896	1.02476034589	0.224751904174	0.0157062950709	0.379957566361	0.112598833278	0.0520831221527	0.364417418388	0.0733654501487	0.296429917666	-60.677	-36.332	24.606
39	1.28725178512	1.12610603332	0.276473810923	0.953902719176	0.238284606243	0.0696948016658	0.206548142685	-0.740456823108	0.473917850105	0.324487570473	-0.207392374161	-0.9540550499	0.260639646552	0.867811797355	-0.0629191686565	0.457618486043	0.205477833265	-0.191051761085	2.00036311938	0.0527833659344	1.63392127378	-10.803	-30.202	35.383
40	0.139186433494	-0.280161816747	0.763664426728	3.19916027704	-1.51110268188	4.18039490268	0.503738500382	-0.32438193738	1.22003913453	0.541348860974	-0.770277464409	-1.42103438741	0.541552937403	1.18130964282	0.138860438916	1.07282350135	0.072295005186	-0.285073714459	2.50362876386	-0.19410963683	2.38616691494	6.283	-25.141	47.422
41	0.020826201384	-0.115539694817	0.142609705613	0.404599417073	-0.740128494746	1.21726389087	0.37882833214	0.816232219458	0.612275716222	-0.0145593819087	-0.0472539051995	-0.197421215065	0.419072810042	0.390864446764	-0.0604310352848	0.538203748346	0.0481136008454	0.184909346424	1.46407200521	-0.392231343838	1.19802510317	-34.47	-5.18	65.695
42	6.2505498635	-1.4879554362	-0.334535955538	1.08913274825	1.16487739335	1.97306988399	-0.0939403943125	0.56426763213	-1.20616759117	-0.140695818234	0.138987398468	0.117459309434	0.0511879486511	0.100347995735	-0.035619394318	0.451079706625	-0.0182424033915	-0.0444522363955	0.355931808546	-0.173936179639	0.795654382746	-82.418	-7.924	68.791
43	1.59886905863	-1.05705310454	-1.23112224612	2.75618494802	-0.0895486199329	2.45758324763	0.120009606642	0.0809035957112	-0.221008573375	0.239153280009	0.0988774071458	0.533051073065	0.381903637508	-0.738152295067	-0.128404574768	0.483091595276	-0.186383342263	-0.138908568716	0.359509348817	-0.033312930453	0.629788579112	-94.441	-6.4	79.691
44	2.70582168429	2.61096891182	2.61843033514	7.81228866121	-0.117108250219	5.98675187462	0.380613444579	0.774589894862	-1.71656293629	-1.39763408661	0.637957327857	0.117878039361	0.470246796963	0.200700527734	-0.490631612109	0.569619970974	-0.0574930730474	-0.308650064261	1.07148873889	-0.256999957369	0.794914268932	-101.648	-4.615	64.538
45	2.26503925341	0.118564526071	-0.628682144989	2.52391576716	0.641009430994	2.2722005058	0.256294439081	0.318508958408	-0.796318109637	0.142376141412	-0.452959343856	0.762110310287	0.044294081721	-0.514304303887	0.124640932913	0.328100816797	-0.164065624508	-0.0546110762361	0.41546688352	-0.167991235065	0.636442075358	-99.877	0.943	80.354
46	4.70537712002	0.800153707486	2.92312835953	6.57971372384	2.36495088779	2.64612096705	0.0404278988497	0.671827306141	-2.25142462957	-0.0224174179185	0.324082232566	0.441342983147	0.735841913866	-0.00968943313647	-0.492653857754	0.44310979259	0.0242242967926	-0.0532628027869	0.628469323077	-0.385318962678	1.26791815149	-67.795	-12.455	66.354
47	1.9470302038	-0.787544414432	-0.695844329337	0.360073252711	0.301686850158	0.24432433865	0.236173221648	0.539787045119	-0.61816644048	-0.0597988986731	0.144887080859	-0.475053980477	0.1852709824	0.979063419024	-0.0151028634814	0.40693336602	0.189652573298	0.0365807221139	1.40429926026	-0.614959890386	1.16765843737	-38.536	-8.54	69.344
48	6.5977027719	1.97657475502	-0.0544628452219	1.73007846967	-1.5592303309	5.91154099942	0.384611381479	-2.10427520211	0.288590994673	0.171552287086	0.237318928489	0.170799007679	-0.277114479494	0.663837927789	-0.539008831324	0.577732485011	0.0877721995369	0.004602445506	0.925992256577	-0.413627693936	1.25969887617	-46.175	-8.42	80.382
49	3.08828356869	-2.97850323546	-0.514375212145	3.23381909742	0.322626185406	0.933321647699	0.103778149981	0.402008885985	0.0721057978227	-0.384202998426	0.144126731721	-0.626877813722	0.168745691082	0.909616338256	0.0604766435785	0.282867324951	0.0583760148226	0.0868579998179	1.00304539261	-0.222756820003	0.646913988647	-49.084	4.316	76.586
50	0.236960528497	0.82818417952	-0.473229039908	2.99991424951	-2.02363332308	3.15113994682	0.0740173735405	0.399016025208	-2.48852484131	0.792628979616	-0.66087793579	1.20288748698	0.106746179315	-0.443631470602	0.492110926099	0.917451617454	0.260590911012	0.284867985161	1.49831488897	-0.284429347847	2.56767342531	-4.388	-15.958	74.867
51	6.93295089607	2.62483952504	3.16688009357	1.12875537866	0.885586660264	2.05245109913	0.142074636262	-0.317104755283	0.707347783154	0.127762941778	0.12248540547	-0.793218584259	-0.252416895706	-0.13545340784	-0.0936889368091	0.495707024669	-0.146724211795	-0.033083192862	1.22522056123	-0.294080882772	1.18530496363	-25.801	-11.757	45.299
52	2.5976884868	2.25535257384	0.784759433636	1.98672170296	0.61297991344	1.49522164394	0.148846806761	-0.481549926071	1.40217935139	0.373854737617	0.227447720138	0.112698976279	-0.248596275173	0.230786844048	-0.115449658702	0.600939740189	-0.0628048816461	0.152470154819	0.435755531205	-0.36065511162	1.0321302119	-73.582	-6.422	41.152
53	4.27399395474	-3.95317600357	-0.464581501762	8.36559606088	-5.17395776184	6.73207686337	0.00295760278474	-0.055596744202	-0.850253628273	0.168111369405	0.172413607916	2.12242452508	1.09024530393	-0.0450605559783	0.438884655809	0.660168435674	0.151462501754	0.132078550684	0.258106929265	-0.435084498703	1.12327965669	-71.655	-14.282	46.069
54	2.28327773235	-0.972105085295	0.221184107045	2.90185026677	0.577449878423	2.69663737024	0.244640880823	-0.20301545474	0.461722119672	0.123558756583	-0.20552041464	0.797692430397	-0.0860963285257	-0.705839984926	0.0217649741602	0.395358802811	0.067290110222	0.210039685152	0.446511940326	-0.113722996218	0.72169209903	-92.732	-17.472	33.339
55	5.97273348762	-0.578412781477	0.276215592608	0.634905783576	0.733366946309	2.38692391925	-0.184608912974	0.0894556766897	1.45807981312	-0.164282696701	0.0862335813161	-0.0485685270854	-0.986677850039	0.152035240962	-0.416297473337	0.511813086031	0.109681837618	0.165546622592	0.45163898272	-0.188852828044	0.83528852999	-75.068	-9.412	33.997
56	5.11225741577	1.94745928934	0.848401297121	0.966033792535	0.271608291066	0.178441285763	-0.179337397132	0.0887144480005	0.805512706093	0.0951758075726	0.225546593684	-0.439457446495	-0.055408309973	0.888048485737	0.222091014559	0.439928718237	-0.209594812961	0.0113318888413	1.30663163964	-0.439728647202	1.24414250023	-30.901	-8.419	38.93
57	2.03824903571	-1.16317041934	4.28393653815	3.0386883592	-2.29765741672	9.00846710843	-0.604003897041	1.89623339885	1.04901787022	0.653144046159	0.391744935627	0.233332681978	-0.235489525124	1.11316250999	0.232017768413	0.749487003285	-0.267212002822	0.0593277275809	0.821357616885	0.212914370856	1.11008882484	-53.624	-12.919	30.965
58	4.91372174427	3.92360691669	0.221828608825	3.78406240433	0.142184968459	0.793729801926	0.137171866385	-0.482595334143	-0.221697337714	0.292263912092	0.087076953137	-0.871697360961	-0.00563432517367	0.893496548224	0.0656487216629	0.361141748158	-0.0748789534441	-0.0153848039923	0.997499391308	-0.185049767183	0.586158456238	-41.798	-21.877	34.416
59	1.23209655589	-1.6299818337	0.887358889373	3.83060571362	0.126398308951	1.63391455831	0.601677536241	-0.199073849775	1.12558506957	0.353751545613	-1.22509289139	-0.859869389648	-0.63970774599	1.6301697909	0.32909190405	0.802190921248	-0.428275897362	-0.0630185366935	1.9277742546	-0.0898843303428	1.65616577233	2.644	-6.711	38.09
60	0.0608291351511	-0.0242209233995	-0.00511096663955	0.0906178693626	0.0486715602262	0.055490400159	-1.01720053946	1.27250711777	0.393455061184	-0.154659877327	0.212763788926	0.483244272603	0.951927329199	-1.37180926154	0.715410040422	1.34196936555	-0.599846952883	0.477976064674	2.1508343644	-0.2820088044	1.72922358568	3.036	2.654	34.371

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	( CHAIN A AND RESID 0:104 )	A	0 - 104
2	X-RAY DIFFRACTION	2	( CHAIN A AND RESID 105:186 )	A	105 - 186
3	X-RAY DIFFRACTION	3	( CHAIN A AND RESID 187:213 )	A	187 - 213
4	X-RAY DIFFRACTION	4	( CHAIN A AND RESID 214:272 )	A	214 - 272
5	X-RAY DIFFRACTION	5	( CHAIN A AND RESID 273:321 )	A	273 - 321
6	X-RAY DIFFRACTION	6	( CHAIN A AND RESID 322:381 )	A	322 - 381
7	X-RAY DIFFRACTION	7	( CHAIN A AND RESID 382:389 )	A	382 - 389
8	X-RAY DIFFRACTION	8	( CHAIN A AND RESID 390:432 )	A	390 - 432
9	X-RAY DIFFRACTION	9	( CHAIN A AND RESID 433:456 )	A	433 - 456
10	X-RAY DIFFRACTION	10	( CHAIN A AND RESID 457:494 )	A	457 - 494
11	X-RAY DIFFRACTION	11	( CHAIN B AND RESID 0:81 )	B	0 - 81
12	X-RAY DIFFRACTION	12	( CHAIN B AND RESID 82:193 )	B	82 - 193
13	X-RAY DIFFRACTION	13	( CHAIN B AND RESID 194:212 )	B	194 - 212
14	X-RAY DIFFRACTION	14	( CHAIN B AND RESID 213:217 )	B	213 - 217
15	X-RAY DIFFRACTION	15	( CHAIN B AND RESID 218:264 )	B	218 - 264
16	X-RAY DIFFRACTION	16	( CHAIN B AND RESID 265:338 )	B	265 - 338
17	X-RAY DIFFRACTION	17	( CHAIN B AND RESID 339:371 )	B	339 - 371
18	X-RAY DIFFRACTION	18	( CHAIN B AND RESID 372:387 )	B	372 - 387
19	X-RAY DIFFRACTION	19	( CHAIN B AND RESID 388:476 )	B	388 - 476
20	X-RAY DIFFRACTION	20	( CHAIN B AND RESID 477:493 )	B	477 - 493
21	X-RAY DIFFRACTION	21	( CHAIN C AND RESID 0:83 )	C	0 - 83
22	X-RAY DIFFRACTION	22	( CHAIN C AND RESID 84:191 )	C	84 - 191
23	X-RAY DIFFRACTION	23	( CHAIN C AND RESID 192:213 )	C	192 - 213
24	X-RAY DIFFRACTION	24	( CHAIN C AND RESID 214:264 )	C	214 - 264
25	X-RAY DIFFRACTION	25	( CHAIN C AND RESID 265:322 )	C	265 - 322
26	X-RAY DIFFRACTION	26	( CHAIN C AND RESID 323:352 )	C	323 - 352
27	X-RAY DIFFRACTION	27	( CHAIN C AND RESID 353:362 )	C	353 - 362
28	X-RAY DIFFRACTION	28	( CHAIN C AND RESID 363:393 )	C	363 - 393
29	X-RAY DIFFRACTION	29	( CHAIN C AND RESID 394:484 )	C	394 - 484
30	X-RAY DIFFRACTION	30	( CHAIN C AND RESID 485:493 )	C	485 - 493
31	X-RAY DIFFRACTION	31	( CHAIN D AND RESID 4:66 )	D	4 - 66
32	X-RAY DIFFRACTION	32	( CHAIN D AND RESID 67:94 )	D	67 - 94
33	X-RAY DIFFRACTION	33	( CHAIN D AND RESID 95:266 )	D	95 - 266
34	X-RAY DIFFRACTION	34	( CHAIN D AND RESID 267:317 )	D	267 - 317
35	X-RAY DIFFRACTION	35	( CHAIN D AND RESID 318:361 )	D	318 - 361
36	X-RAY DIFFRACTION	36	( CHAIN D AND RESID 362:369 )	D	362 - 369
37	X-RAY DIFFRACTION	37	( CHAIN D AND RESID 370:390 )	D	370 - 390
38	X-RAY DIFFRACTION	38	( CHAIN D AND RESID 391:418 )	D	391 - 418
39	X-RAY DIFFRACTION	39	( CHAIN D AND RESID 419:479 )	D	419 - 479
40	X-RAY DIFFRACTION	40	( CHAIN D AND RESID 480:490 )	D	480 - 490
41	X-RAY DIFFRACTION	41	( CHAIN E AND RESID 2:36 )	E	2 - 36
42	X-RAY DIFFRACTION	42	( CHAIN E AND RESID 37:94 )	E	37 - 94
43	X-RAY DIFFRACTION	43	( CHAIN E AND RESID 95:129 )	E	95 - 129
44	X-RAY DIFFRACTION	44	( CHAIN E AND RESID 130:146 )	E	130 - 146
45	X-RAY DIFFRACTION	45	( CHAIN E AND RESID 147:268 )	E	147 - 268
46	X-RAY DIFFRACTION	46	( CHAIN E AND RESID 269:285 )	E	269 - 285
47	X-RAY DIFFRACTION	47	( CHAIN E AND RESID 286:368 )	E	286 - 368
48	X-RAY DIFFRACTION	48	( CHAIN E AND RESID 369:393 )	E	369 - 393
49	X-RAY DIFFRACTION	49	( CHAIN E AND RESID 394:449 )	E	394 - 449
50	X-RAY DIFFRACTION	50	( CHAIN E AND RESID 450:495 )	E	450 - 495
51	X-RAY DIFFRACTION	51	( CHAIN F AND RESID 0:36 )	F	0 - 36
52	X-RAY DIFFRACTION	52	( CHAIN F AND RESID 37:78 )	F	37 - 78
53	X-RAY DIFFRACTION	53	( CHAIN F AND RESID 79:85 )	F	79 - 85
54	X-RAY DIFFRACTION	54	( CHAIN F AND RESID 86:234 )	F	86 - 234
55	X-RAY DIFFRACTION	55	( CHAIN F AND RESID 235:288 )	F	235 - 288
56	X-RAY DIFFRACTION	56	( CHAIN F AND RESID 289:385 )	F	289 - 385
57	X-RAY DIFFRACTION	57	( CHAIN F AND RESID 386:393 )	F	386 - 393
58	X-RAY DIFFRACTION	58	( CHAIN F AND RESID 394:449 )	F	394 - 449
59	X-RAY DIFFRACTION	59	( CHAIN F AND RESID 450:470 )	F	450 - 470
60	X-RAY DIFFRACTION	60	( CHAIN F AND RESID 471:494 )	F	471 - 494