Terminal Hinge

Inner Loop C-Terminal Hinge

Number of Sequences

PVW AIGTG KTA 92

PIW AIGTG KSA 58

PVW AIGTG KVA 58

PIW AIGTG KTA 57

PVW AIGTG KSA 46

PIW AIGTG KSS 42

PIW AIGTG KTC 19

PLW AVGTG KSA 18

PVW AIGTG RVA 18

PVW AIGTG LTA 15

PVW AIGTG RTA 13

PVW AIGTG LTP 12

PIW AIGTG KAA 11

PVW AIGTG LAA 9

PVW AIGTG VVA 9

PIW AIGTG DTC 8

PIW AIGTG RTA 6

PIW AIGTK KSA 6

PVW AIGTG KVP 6

PIW AIGTG KVA 4

PVW AIGTG ETA 4

PVW AIGTG KNA 4

PVW AIGTG RHA 4

PVW AIGTG RTP 4

PIW AIGTG KNA 3

PIY SIGTG VSA 3

PLW AIGTG KTA 3

PVW AIGTG DTA 3

PVW AIGTG MVA 3

PEW AIGKA SSA 2

PIW AIGTD LEL 2

PIW AIGTG CTA 2

PIW AIGTG HVP 2

PIW AIGTG VSA 2

PSS AINSG NCA 2

PVW AIGTG HSA 2

PVW AIGTG KVC 2

PVW SIGTG VVA 2

! YC!

PEW AIGKA NSA 1

PIW AIATG KSA 1

PIW AIGSG ASA 1

PIW AIGTG DTA 1

PIW AIGTG ESA 1

PIW AIGTG ETA 1

PIW AIGTG ETC 1

PIW AIGTG HSA 1

PIW AIGTG LIP 1

PIW AIGTG LTP 1

PIW AIGTG LVA 1

PIW AIGTG NVP 1

PIW AIGTG QTC 1

PIW AIGTG RSA 1

PIW AIGTG RTP 1

PIW AIGTG VTA 1

PIW AIGTG VTP 1

PIW SIGSD MIP 1

PIW SIGSG NSA 1

PIW SIGTG VSA 1

PKW AIGTN TIP 1

PLW AIGTG RTA 1

PRW AIGTG KTP 1

PSW AIGSG TSA 1

PTW AIGKK DSA 1

PVW AIGKP QPA 1

PVW AIGNG QNA 1

PVW AIGSG KAA 1

PVW AIGSG LTA 1

PVW AIGSG NPA 1

PVW AIGSG QAA 1

PVW AIGSG SSA 1

PVW AIGSN TIP 1

PVW AIGTG AVA 1

PVW AIGTG EVA 1

PVW AIGTG ITA 1

PVW AIGTG KAA 1

PVW AIGTG KSS 1

PVW AIGTG LSA 1

PVW AIGTG LVP 1

PVW AIGTG NSP 1

PVW AIGTG NTA 1

PVW AIGTG QTA 1

! YT!

PVW AIGTG RNA 1

PVW AIGTG RNC 1

PVW AIGTG RSA 1

PVW AIGTG RVP 1

PVW AIGTG TPA 1

PVW AIGTG VAA 1

PVW AIGTG VTA 1

PVW AIGTN RTA 1

PVW AIGTN VTA 1

PVW AVGTG NTA 1

PVW AVGTG RSA 1

PVW SIGSC MVP 1

PVW SIGTG ITP 1

! YW!

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Library Average number of mutations per design

Percent of mutants that complement TIM- deficient strain

Closed 2.88 100

Closed biased 3.67 100

Even 4.00 95.83

Open biased 4.38 66.67

Open 4.33 70.83

! YX!

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&7(/G/(8J!

Hinge sequences

Percent WT activity

In vivo complementation

assay

Apparent Tm (˚C)

MD metric

PIWVTA 96.89 + 50 0.999

PIWITA 75.90 + 50 1.000

PIWKTA 71.57 + 50 0.994

PIWLTA 74.58 + 50.3 0.982

PIWTTA 80.24 + 50 0.996

PIWVVA 84.48 + 50 0.921

PQWVTA 27.73 + 46 0.987

PIWRTA 67.36 + 50 0.656

PIWQTA 94.28 + 50 0.998

PVWVTA 22.39 + 44 0.974

PMWVTA 32.45 + 47 0.205

PLWVTA 29.41 + 47 0.846

PIWETA 49.85 + 51 0.993

PRWVTA 46.51 + 51 0.258

PIWYTA 85.14 + 50.7 1.000

PIWFTA 98.39 + 50 0.995

PEWVTA 76.67 + 50 1.000

PYWVTA 20.91 + 44 0.818

PIWVSA 60.47 + 50 0.867

PAWVTA 45.74 + 50 0.382

PIWVAA 43.91 + 50 0.995

PFWVTA 43.07 + 50 0.820

PIWNTA 47.10 + 50 0.988

PIWSTA 86.76 + 49.7 0.998

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Hinge sequences

Percent WT activity

In vivo complementation

assay

Apparent Tm (˚C)

MD metric

PIFQTA 58.92 + 49 0.087

PIFKTA 60.77 + 49.3 0.998

PIFRTA 2.38 + 51 0.953

PMFQTA 15.53 + 51 0.967

PIFQVA 29.15 + 48 0.333

PQFQTA 38.36 + 48 0.951

PIFETA 30.10 + 48 0.951

PVFQTA 43.78 + 49 0.054

PIFTTA 44.88 + 48.7 0.164

PIHQTA 26.62 + 48.7 0.988

PLFQTA 53.95 + 50 0.999

PEFQTA 2.38 + 45.7 0.986

PIWKTG 55.08 + 49 0.758

PIFLTA 50.42 + 50 0.994

PRFQTA 26.01 + 50 0.949

PIWQTG 37.68 + 49 0.994

PIYKTA 89.37 + 50 0.964

PMWKTG 35.85 + 47 0.249

PAFQTA 2.38 + 51 0.134

PYFQTA 2.38 + 49 1.000

PVWKTG 43.00 + 50 0.981

PIFQSA 58.21 + 49 0.130

PIFMTA 35.19 + 46 0.999

PLWKTG 2.80 + 47 0.432

! YO!

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Hinge sequences

Percent WT activity

In vivo complementation

assay

Apparent Tm (˚C)

MD metric

PMFQTA 15.53 + 51 0.967

PMFQVA 20.39 + 51 0.587

PMFKTA 16.00 + 47 0.999

PMHQTA 24.45 + 48.7 0.993

PEFQTA 2.38 + 45.7 0.986

PIFQTA 58.92 + 49 0.087

PVFQTA 43.78 + 49 0.054

PMFETA 13.94 + 49 0.518

PMHQTN 28.84 + 48.7 0.980

PMFQTS 11.09 + 48 0.998

PMFRTA 11.58 + 48.3 0.730

PQFQTA 38.36 + 48 0.951

PLFQTA 53.95 + 50 0.999

PAFQTA 2.38 + 51 0.134

PRFQTA 26.01 + 50 0.949

PMFQSA 34.25 + 51 1.000

PMFQIA 17.02 + 48.7 0.999

PMHQTD 6.82 - 47.7 0.219

PMFMTA 13.50 + 52 0.137

PDFQTA 15.20 + 49 0.115

PMFQYA 7.52 + 50 0.995

PYFQTA 2.38 + 49 1.000

PMFQAA 34.92 + 50.7 0.977

PKFQTA 19.06 + 50.7 0.989

! YY!

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Hinge sequences

Percent WT activity

In vivo complementation

assay

Apparent Tm (˚C)

MD metric

PMHQVN 2.38 - 51 0.205

PMFQVA 20.39 + 51 0.587

PMHQTN 28.84 + 48.7 0.980

PMFQVS 10.81 + 48.7 0.996

PMHQIN 2.38 - 47 0.894

PMHQVD 4.32 + 48 0.994

PMHQYN 2.38 + 47.7 0.306

PMHKTN 9.96 + 48 0.991

PEHQVN 2.38 + 45.7 0.651

PMHQFN 2.38 + 47.7 0.951

PMHETN 2.38 + 51 0.989

PMHQSN 8.35 + 48 0.995

PMFQSA 34.25 + 51 1.000

PVHQVN 4.24 - 50 0.978

PMHQQN 2.38 - 46.7 0.993

PAHQVN 2.38 - 50 0.998

PMHQKN 2.38 + 47 0.254

PMHQEN 2.38 - 47 0.511

PMHQNN 2.38 + 47 0.998

PMHQRN 4.79 + 51 0.419

PMHMTN 2.38 + 46.3 0.174

PMFQRA 8.32 + 48 0.358

PDHQVN 5.58 - 47.7 0.407

PMHQVT 2.38 - 51 0.615

! YZ!

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Hinge sequences

Percent WT activity

In vivo complementation

assay

Apparent Tm (˚C)

MD metric

PMHQVN 2.38 - 51 0.205

PMHQTN 28.84 + 48.7 0.980

PMHQVT 2.38 - 51 0.615

PMHQIN 2.38 - 47 0.894

PMFQVA 20.39 + 51 0.587

PMAQVT 2.38 + 47 0.987

PMSQVT 2.38 + 47.3 0.709

PMHQYN 2.38 + 47.7 0.306

PMFQVS 10.81 + 48.7 0.996

PMAQTT 2.38 + 47.3 0.931

PMHQFN 2.38 + 47.7 0.951

PMHQVD 4.32 + 48 0.994

PMHQLN 2.38 + 47 0.028

PMHQFT 2.45 - 51 0.987

PMEQVT 5.67 + 48 0.998

PMHQLT 2.38 + 47 0.103

PEHQVN 2.38 + 45.7 0.651

PMHKVN 9.28 + 48 0.052

PMHQQN 2.38 - 46.7 0.993

PEHQVT 2.38 + 44.3 0.312

PMHQKN 2.38 + 47 0.254

PMHKVT 2.38 + 48 0.816

PMHQEN 2.38 - 47 0.511

PMAQLT 2.38 - 48 0.999

! Y[!

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Most active hinges

Activity Library

Natural double

hinge mutant?

Natural N-terminal

hinge?

Natural C- terminal

hinge?

PIWFTA 98.4 Closed no yes no A

PIWVTA 96.9 Closed yes yes yes A

PIWQTA 94.3 Closed no yes yes A

PIYKTA 89.4 Closed

Biased no yes yes A

PIWSTA 86.8 Closed no yes no A

PIWYTA 85.1 Closed no yes no A

PIWVVA 84.5 Closed no yes yes A

PIWTTA 80.2 Closed no yes no A

PEWVTA 76.7 Closed no yes yes A

PIWITA 75.9 Closed no yes yes A

PIWLTA 74.6 Closed no yes yes A

PIWKTA 71.6 Closed yes yes yes A

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CPD alone

After MD screening

# true positive designs 58 37

# true negative designs NA 20

# false positive designs 38 18

# false negative designs NA 21

# designs to characterize 96 55

# designs to discard NA 41

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